text:
A Disease-causing Mutation in the Active Site of Serine Palmitoyltransferase Causes Catalytic Promiscuity*

The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has ∼10–20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype.

Serine palmitoyltransferase (SPT) 3 catalyzes the committed and rate-limiting step in sphingolipid biosynthesis. Until recently, it was believed that there was a single isozyme of this enzyme containing two subunits, LCB1 and LCB2, with the catalytic domain located at the interface and composed of residues from each subunit. However, Hornemann et al. showed that there was a second isoform of LCB2 (1). More recently, we identified two highly related isoforms of a third subunit, ssSPTa and ssSPTb, which are essential for maximal enzymatic activity (2). Thus, there are at least four potential SPT isozymes, each containing a common hLCB1 subunit and one of each of the two hLCB2 and ssSPT subunits. Examination of the acyl-CoA preferences of the hLCB1/LCB2a and hLCB1/LCB2b heterodimers and the hLCB1/LCB2/ssSPT heterotrimers showed that both the LCB2 isoform and the ssSPT isoform conferred different acyl-CoA preferences (2,3), revealing a previously unappreciated complexity in SPT substrate selectivity, and the corresponding long chain base (LCB) products.
Mutations in the hLCB1 subunit of SPT have been demonstrated to cause the autosomal dominant late-onset hereditary sensory neuropathy, type I (HSAN1) (4 -6). In addition, an HSAN1-like phenotype is seen in transgenic mice overexpressing the LCB1 C133W mutation (7). Because modeling studies placed the Cys-133 residue near the active site (8 -10), reduced SPT activity was seen in transgenic mice (7), and in vitro characterization indicated that the mutant heterodimers were catalytically inactive (7,8), it appeared that the HSAN1 phenotype might be the result of haploinsufficiency. However, several lines of evidence suggest that this is not the case. First, measurement of SPT activity in lymphoblastoid cell lines established from normal and affected members of HSAN1 families showed a wide range of SPT activities with some unaffected individuals having less SPT activity than some of the affected relatives (11). Second, despite the fact that heterozygous mLCB1 knock-out mice have a greater reduction in SPT activity than the transgenic mice overexpressing the LCB1 C133W allele, they show no neurologic impairment (12). Third, the presence of deoxy-LCBs has been reported in both the serum of affected patients (13) and the transgenic mice expressing the mutant allele (12) suggesting that the mutant enzyme is active but has acquired the ability to condense alanine and glycine with acyl-CoAs. Thus, the disease-causing HSAN mutations may result in a gain-of-function. This hypothesis is entirely consistent with the fact that, although many mutations in hLCB1 would inactivate the subunit, only a few mutations have been identified in various HSAN1 kindreds (14). In addition, because transgenic mice co-overexpressing both mutant and wild-type LCB1 have no obvious phenotype (12), it does not appear that disease-causing mutations lead to misfolding of LCB1 resulting in a toxic protein analogous to those responsible for other neurodegenerative diseases (15,16).
The most common HSAN1 mutation resides in a highly conserved domain. Previous studies of yeast SPT demonstrated that this mutation rendered it catalytically inactive (8). The same result was obtained when Chinese hamster LCB1 C133W was cotransfected with LCB2a in CHO cells (7). However, the lack of activity of the mammalian LCB1 133W /LCB2a heterodimers might simply reflect an extremely reduced basal activity of the mutant enzymes that lack an ssSPT subunit. Our recent discovery of the ssSPT subunits and our ability to express the human isozymes in yeast cells lacking endogenous SPT (2) has allowed us to directly test the possibility that the trimeric human SPT isozymes containing the most common HSAN1 mutant, hLCB1 C133W , are in fact enzymatically active and capable of utilizing alanine to synthesize the deoxy-longchain base, 1-deoxySa. Our results demonstrate that this is indeed the case and provide additional evidence that these unusual products may be causally related to the loss of neurons in the dorsal root ganglia of patients with HSAN1.
Plasmids-Construction of plasmids used to express the human SPT subunits in yeast and mammalian cells has been previously described (2). The C133W mutation was introduced into the yeast hLCB1 expression plasmid using QuikChange mutagenesis (Stratagene, La Jolla, CA), and the AvrII restriction fragment from this plasmid was substituted for the corresponding fragment in the mammalian expression plasmid. Yeast plasmids expressing HA-ssSPTa and HA-ssSPTb were constructed by PCR amplifying the ssSPTa or ssSPTb open reading frame to generate a fragment flanked by NheI-XbaI sites, which was ligated into pGH316 (2), followed by insertion of an SpeI-ended HA epitope. The plasmid expressing the hLCB2a-ssSPTa-LCB1 triple fusion protein was constructed by ligating an XbaI-NheI-ended PCR fragment containing hLCB2a into the NheI site of pGH316, followed by insertion of an NheI-XbaI-ended PCR fragment containing hLCB1 into the NheI site to create an hLCB2a-hLCB1 fusion with an in-frame NheI site at the junction. Finally, an XbaI-Nhel-ended ssSPTa PCR fragment was ligated into the Nhe1 between hLCB2a-LCB1 to generate the yeast expression plasmid. To create the mammalian expression plasmid, a BglII/SalI-ended PCR fragment containing hLCB2a-ssSPTa-LCB1 was ligated between the BglII and XhoI sites of pDual GFP-CCM (Vector Biolabs, Philadelphia, PA).
Microsome Preparation and Immunoblotting-Microsomes were prepared from yeast and mammalian cells as previously described (18). hLCB1, hLCB2a, HA-ssSPTa, and HA-ssSPTb were resolved by SDS-PAGE and visualized by immunoblotting using antibodies directed against LCB1, LCB2a, and the HA epitope as previously described (2). GADD153 was detected using the SC-7351 mouse monoclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:250 and glyceraldehyde-3-phosphate dehydrogenase was detected using mouse monoclonal antibody MA B374 (Millipore, Billerica, MA) at a dilution of 1:10,000.
SPT Assays-Microsomal SPT activity was assayed by measuring acyl-CoA-dependent incorporation of [ 3 H]serine into long-chain bases as previously described (2). For determination of kinetic constants, microsomes were incubated for 10 min at the indicated serine or alanine concentrations, with 18 Ci/ml LCB Analysis-LCBs were extracted, derivatized, and analyzed by reversed-phase high-performance liquid chromatography on a C18 column (4 m, Grace Vydac, Hesperia, CA) using an HP Series II 1090 liquid chromatograph with HP Chemstation coupled to an Agilent 1100 series fluorescence detector as previously described (19). The 1-deoxySa standards were either purchased (C18) from Avanti Polar Lipids (Alabaster, Al) or were synthesized (C20) using purified Sphingomonas paucimobilis SPT enzyme (20) with alanine and stearoyl-CoA as substrates to produce C20-3-keto-deoxysphinganine followed by sodium borohydride reduction.

Expression and Activities of Wild-type and Mutant SPT
Heterodimers-It was previously shown that the yeast Lcb1p C180W mutant, which is analogous to the hLCB1 C133W mutant, heterodimerizes with yeast Lcb2p, but that the mutant heterodimers are catalytically inactive (8). To determine whether the hLCB1 C133W mutation abolishes the activity of hLCB1/hLCB2 heterodimers, hLCB1 or hLCB1 C133W was coexpressed with hLCB2a in CHO-LyB cells, and the activities of the human mutant and wild-type heterodimers were compared. Immunoblotting revealed that the hLCB1 C133W mutant and wild-type hLCB1 proteins were expressed at similar levels, and both stabilized cotransfected hLCB2a (Fig. 1A). Because hLCB2a stability is dependent upon its ability to form a heterodimer with hLCB1 (21-22) (Fig. 1A), this result indicates that hLCB1 C133W forms stable heterodimers with hLCB2a. Measurement of SPT activity in microsomes prepared from transfected cells showed that, unlike wild-type hLCB1, which restored some SPT activity that was further increased by the co-expression of hLCB2a, the hLCB1 C133W subunit did not restore SPT activity with endogenous CHO LCB2 or cotransfected hLCB2a (Fig. 1B). Indeed, cotransfection of increasing ratios of the hLCB1 C133W to wild-type hLCB1 showed that SPT activity was inversely proportional to the ratio of mutant/wildtype hLCB1 (Fig. 1C). Thus, although wild-type hLCB1 and hLCB1 C133W have similar affinities for the hLCB2a subunit, the mutant hLCB1 subunit does not form a catalytically active heterodimer with hLCB2a, and behaves as a classic dominant negative mutant.
Expression and Activity of Mutant SPT Heterotrimers-Because even wild-type human SPT heterodimers have only modest activity in yeast and transfected CHO-LyB cells (2), the possibility that the disease-causing mutation simply reduces SPT activity below the level of detection could not be excluded. We therefore investigated the effect of the highly homologous small proteins (ssSPTa and ssSPTb) recently shown to increase SPT activity 50-to 100-fold (2) on the activity of the hLCB1 C133W / LCB2 mutant heterodimers. For these experiments, we took advantage of the ability to express active human SPT isozymes in yeast cells lacking any endogenous SPT activity. As previously reported (2), coexpression of ssSPTa or ssSPTb with hLCB1 and hLCB2a resulted in sufficient increase in activity of the wild-type heterodimer to support growth of the lcb1⌬lcb2⌬ mutant at 37°C (Fig. 2A). More significantly, coexpression of either ssSPTa or ssSPTb with hLCB1 C133W /LCB2a also provided sufficient SPT activity to support growth at both 26°C and 37°C ( Fig. 2A). Measurement of SPT activity in microsomes prepared from yeast cells expressing mutant isozymes containing hLCB2a with either ssSPTa or ssSPTb showed that the mutant enzymes had 10 -20% of wild-type activity (Fig. 2C). This is not a result of differential expression of wild-type and mutant hLCB1 or differential stabilization of hLCB2a, because both mutant and wild-type proteins were expressed to the same level, and equivalent amounts of hLCB2a were seen in the presence of either hLCB1 or hLCB1 C133W (Fig. 2B). A similar activation of hLCB1 C133W /LCB2a by ssSPTa or ssSPTb was also observed in CHO-LyB cells (Fig. 2E). Thus, the C133W diseasecausing mutant forms a catalytically active enzyme when either of the small subunits is present.
Altered Substrate Specificity of Mutant SPT Isozyme-We have recently shown that SPT isozymes containing ssSPTb have an increased ability to condense stearoyl-CoA with serine (2). In addition, although serine is its preferred amino acid substrate, SPT can also condense alanine or glycine with palmitoyl-CoA to generate 1-deoxysphinganine (1-deoxySa) and 1-amino-2-deoxy-n-heptadecane (23)(24). Furthermore, the presence of these LCB species has been reported in HEK 293 cells expressing hLCB1 C133W in Epstein-Barr virus-transformed lymphoblastoid cell lines and plasma derived from HSAN patients (13), and in transgenic mice expressing CHO LCB1 C133W (12). To FIGURE 1. Activity of SPT containing the hLCB1 C133W mutant subunit. hLCB1 or hLCB1 C133W were expressed with or without hLCB2a in CHO-LyB cells, and microsomes were prepared for immunoblotting to ascertain the expression of transfected genes (A) or used to assay SPT activity (B). Alternatively, a constant amount of hLCB1 (0.25 g) was expressed with increasing amounts of hLCB1 C133W and microsomal SPT activity was determined (C). determine whether SPT heterotrimers containing hLCB1 C133W have an increased ability to utilize alanine, wild-type and mutant heterotrimers were expressed in yeast, and formation of C18-1-deoxySa and C20-1-deoxySa was determined. The results showed that wild-type SPT isozymes synthesized neither C18-1-deoxySa nor C20-1-deoxySa at detectable levels. In contrast, yeast cells expressing the hLCB1 C133W mutant along with hLCB2a and ssSPTa synthesized significant amounts of C18-1-deoxySa, but no C20-1-deoxySa (Fig. 3A), whereas cells expressing the hLCB1 C133W and hLCB2a along with ssSPTb produced both C18-1-deoxySa and C20-1-deoxySa (Fig. 3B). As we have previously shown that ssSPTa confers a preference for C16-CoA, whereas ssSPTb confers a preference for C18-CoA (2), we conclude that the C133W mutation allows the mutant SPT isozymes to react with alanine without altering their acyl-CoA preferences. Cells expressing the SPT isozymes containing hLCB2b instead of hLCB2a also accumulated 1-de-oxySa. 4 It is also interesting to note that, although the products of SPT are 3-ketosphinganine or 3-ketodeoxysphinganine, it appears that both are efficiently reduced by Tsc10p and FVT1 in yeast and mammalian cells, respectively, because the 3-keto products were not detected. Similar results were obtained when the mutant SPT isozymes were expressed in CHO-LyB cells. Long-chain base analysis of CHO-LyB cells expressing SPT isozymes containing mutant hLCB1 C133W revealed the presence of substantial amounts of C18-1-deoxySa not found in cells expressing any wild-type isozyme (Fig. 3C). In addition, in contrast to the hLCB1/LCB2a/ ssSPTa heterotrimers, expression of mutant hLCB1 C133W / LCB2a/ssSPTb heterotrimers also produced low but detectable amounts of C20-1-deoxySa (Fig. 3D). Taken together, these results indicate that the mutant heterotrimeric SPT enzymes are active in both yeast and mammalian cells and have an enhanced ability to condense alanine with the preferred acyl-CoA.
Kinetic Properties of the Mutant SPT-To investigate the effect of the HSAN1 mutation on amino acid selectivity, microsomes prepared from yeast expressing the wild-type hLCB1/ hLCB2a/ssSPTa and mutant hLCB1 C133W /hLCB2a/ssSPTa heterotrimeric SPT isozymes were used to measure the K m and V max for serine. The results showed that the wild-type enzyme had a K m for serine of ϳ0.75 mM and a V max of ϳ1350 pmol/ mg/min (Fig. 4A). By comparison the K m and V max for serine of the mutant enzyme were ϳ1.4 mM and ϳ275 pmol/mg/min, respectively (Fig. 4B). Thus, the major affect of the HSAN1 mutation is on V max . With alanine as substrate, the HSAN1 mutant enzyme had a K m of ϳ9.6 mM and a V max of ϳ110 pmol/mg/min (Fig. 4C). The wild-type SPT did not efficiently utilize alanine, precluding the direct measurement of a K m . However, inhibition by alanine of serine utilization by the wildtype and mutant enzymes revealed a similar relative affinity for alanine with K i values of 2 mM and 5 mM, respectively (Fig. 4D). Combined with the similar affinities of the mutant and wildtype enzymes for serine and the significant rate of reaction of the mutant enzyme with alanine, these data suggest that the major effect of the HSAN1 mutation is to perturb the active site in such a manner as to allow bound alanine to be activated to react with the acyl-CoA substrate.
Effects of Varying Extracellular Alanine and Serine on 1-DeoxySa Accumulation-Because the normal extracellular concentrations of serine and alanine are well below the K m values of SPT, the possibility that altering the ratio of serine to alanine would affect the amount of 1-deoxySa synthesized was investigated. It is clear from the data in Figs. 3 and 5B that yeast and mammalian cells expressing the mutant HSAN1 enzyme produce a small but significant amount of 1-deoxySa even when no additional alanine is added to the growth medium. Based on the serum concentration of alanine, ϳ0.12 mM alanine was present in the growth medium of the CHO-LyB cells. Addition of as little as 1 mM alanine elicited a large increase in 1-deoxySa production (Fig. 5C). Perhaps more importantly, increasing the serine concentration from 0.12 mM in normal medium to 1.12 mM reversed the effect of added alanine (Fig. 5D), and addition of 10 mM serine completely abolished 1-deoxySa synthesis (Fig.  5E). Thus, relatively small increases in the ratio of serine to alanine have the potential to inhibit 1-deoxySa synthesis by the HSAN1 enzyme containing the C133W mutation in hLCB1.

1-DeoxySa Induces ER Stress
Response-1-deoxySa has been implicated as the potentially neurotoxic agent in HSAN1 (12)(13). However, the basis for this toxicity has not been deter-  mined. Because 1-deoxySa is synthesized in the ER, we examined the possibility that it induces ER stress. Initial experiments expressing the three SPT subunits from three cotransfected expression plasmids provided some indication that the HSAN1 mutant heterotrimer increased GADD153 immunoreactivity, a marker for ER stress and the unfolded protein response (25). However, even the wild-type enzyme, though to a lesser extent, also increased GADD153 expression (data not shown). This is likely due to non-stoichiometric expression of the three subunits and the consequent misfolding of monomeric subunits. To obtain stoichiometric expression, we extended our previous observation that a functional SPT enzyme could be synthesized as an LCB2-LCB1 fusion protein (26) and created a triple fusion containing all three subunits (Fig. 6A). Their arrangement was dictated by the observations that : 1) the C terminus of LCB2 is cytoplasmic 5 ; 2) split ubiquitin two-hybrid experiments place the N termini of the ssSPTs in the cytoplasm and the ssSPTs contain a single predicted transmembrane domain; and 3) the N terminus of LCB1 is luminal and the C terminus is cytoplasmic (18). This arrangement is analogous to the known organization of the coccolithoviral single-chain SPT, in which the Lcb2-like domain precedes the Lcb1-like domain with the two separated by a short spacer (26). Thus, we reasoned that an hLCB2-ssSPT-LCB1 triple fusion protein would adopt a topology that recapitulates that of the native SPT heterotrimer.
Indeed, the triple fusion protein had activity equal or greater to, and biochemical properties indistinguishable from, the normal heterotrimer. 5 In addition, introduction of the HSAN1 mutation into the triple fusion results in 1-deoxySa synthesis comparable to that obtained by coexpression of mutant hLCB1 C133W , hLCB2a, and ssSPTa (compare Figs. 5B and 6B).
Using the triple fusion protein, it is clear that the wild-type enzyme has virtually no affect on GADD153 expression, whereas the mutant fusion protein significantly elevates GADD153 immunoreactivity (Fig. 6C). This increase is greater when cells are grown in 10 mM alanine and is blocked by inclusion of 10 mM serine (Fig. 6C), suggesting that 1-deoxySa, or a metabolite, induces ER stress. Indeed, the level of GADD153 was substantially elevated in cells grown in 1 M deoxySa (Fig.  6D). Thus, there is a clear correlation between 1-deoxySa production and the ER stress response.

DISCUSSION
Until recently, SPT was thought to be a simple heterodimer composed of two similarly sized subunits, hLCB1 and hLCB2, with each subunit contributing residues that form a catalytic site at their interface for the condensation of serine and palmitoyl-CoA. The identification a second isoform (hLCB2b) of the pyridoxal phosphate-binding subunit, LCB2, added some complexity, because this isoform appears to confer a broader acyl-CoA preference facilitating the incorporation of shorter acyl-   CoAs (2,3). This is similar to what was observed for the singlechain SPT we previously characterized from Emiliania huxleyi (26). However, the activity of either heterodimer is negligible in the absence of a third subunit (ssSPT) that increases SPT activ-ity 50-to l00-fold (2). There are two isoforms of this subunit. Both increase SPT activity of hLCB1/hLCB2a and hLCB1/ hLCB2b heterodimers. The hLCB1/hLCB2a/ssSPTa isozyme has a clear preference for C-16 acyl-CoA, and the hLCB1/ hLCB2b/ssSPTa prefers C-14 acyl-CoAs. In contrast, the hLCB1/hLCB2a/ssSPTb and hLCB1/hLCB2b/ssSPTb isozymes have a clear preference for longer rather than shorter acyl-CoAs (2). Indeed, the hLCB1/hLCB2b/ssSPTb isozyme prefers C-18 and C-20 acyl-CoAs. Thus, the acyl-CoA preferences ascribed to the hLCB1/hLCB2 heterodimers are likely a consequence of the relative amounts of ssSPTa and ssSPTb in transfected cells and the complexities introduced by the transient overexpression of individual subunits of a multimeric enzyme.
The activation of the hLCB1/LCB2a heterodimer by the ssSPT subunits allowed us to investigate whether the unusual LCB, 1-deoxySa, suggested to underlie the pathophysiology of HSAN1, is generated by the mutant SPT enzymes containing the C133W mutation in hLCB1. Our results show that, although the mutant heterotrimeric SPTs have the same acyl-CoA preferences as their wild-type counterparts, the mutant enzymes have acquired the ability to activate and condense alanine with acyl-CoAs to produce the 1-deoxySas. Although it has been suggested that the HSAN1 mutations cause a higher affinity for alanine (13), two lines of evidence suggest that the C133W mutation does not significantly alter the amino acid binding site of the mutant SPT enzyme. First, the mutant enzyme has only a 2-fold greater K m for serine than the wildtype enzyme. Second, the K i for alanine inhibition of serine utilization by the mutant enzyme is similar to that of the wildtype SPT. The more likely effect of the mutation is to perturb the catalytic site in such a way as to facilitate the formation of the external aldimine with alanine and/or allow the external aldimine to be more efficiently deprotonated to form and stabilize the reactive quininoid intermediate. This is consistent with the crystal structures of the wild-type and mutant soluble bacterial enzymes, which show that the HSAN1-like mutation alters the relative positions of multiple hydrophobic residues contributed by both subunits at the dimer interface (9). Further studies of the HSAN1-like mutations of the bacterial SPT enzyme are likely to reveal the precise effect of these amino acid substitutions on catalysis.
Interestingly, Hanada et al. (27) reported that alanine did not inhibit SPT purified from CHO cells. In preliminary studies we have observed that the ssSPT subunits dissociate during purification. This raises the possibility that the ssSPT subunit is necessary for alanine binding. Resolution of this question will require the purification of sufficient heterodimeric and heterotrimeric SPT for kinetic and structural studies. The SPT fusion proteins should facilitate these studies.
It has been suggested that 1-deoxySa contributes to the pathophysiology of HSAN1 (12,14). The observation that GADD153, an ER stress response protein, is induced by growth in 1-deoxySa as well as the fact that GADD153 levels are increased in cells expressing the disease-causing SPT isozyme support this hypothesis. The ability of physiological extracellular concentrations of alanine to increase both 1-deoxySa production and the ER stress response, combined with the finding FIGURE 6. 1-deoxySa induces ER stress. A, model of the hLCB2-ssSPT-LCB1 triple fusion SPT in which ssSPT (stippled) is inserted between hLCB2 and hLCB1. Topologies are based on hydropathy analyses as well as preliminary experimental data as described in the text. Cys-133, which when mutated to tryptophan causes HSAN, is indicated (C). The pyridoxal phosphate-binding lysine in hLCB2, which in the crystal structure of S. paucimobilis lies directly across the dimer interface from Cys-133 (9), is also indicated (K). B, CHO-LyB cells expressing mutant human LCB2a-ssSPTa-LCB1 C133W (bottom panel), but not wild-type LCB2-ssSPTa-LCB1 (top panel), fusion SPT accumulate 1-de-oxySa (DoxSA). C, cells transfected with vector (lanes 1-3), or plasmids expressing hLCB2a-ssSPTa-LCB1 (WT; lanes 4 -6), or hLCB2a-ssSPTa-LCB1 C133W (C133W; lanes 7-9) were grown in Ham's F-12 with or without 10 mM serine or 10 mM alanine for 48 h. As controls, untransfected cells were treated with vehicle (lane 10) or 20 g/ml tunicamycin for 5 h (lane 11). LCB2a-ssSPTa-LCB1, GADD153, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were visualized by immunoblotting following fractionation of total cellular proteins by SDS-PAGE. that serine blocks both of these effects, suggests that it may be possible to directly test this hypothesis by modulating the ratios of serine to alanine in the diet of HSAN1 transgenic mice. A positive result would suggest a potentially simple therapeutic strategy for treating affected individuals. A negative result would be harder to interpret. It could indicate that modification of dietary serine does not alter the serine concentration in the DRG neurons that are most affected in HSAN1 patients. Alternatively, it could indicate that the catalytic promiscuity reflects a more subtle alteration in the enzyme, which over time elicits the late onset peripheral neuropathy. It will therefore be important to examine the enzymatic properties of the other HSAN1 disease-causing mutations.


extfieldsofstudy: Biology Medicine

text:
Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

Background Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region. Methodology/Principal Findings Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs. Conclusions/Significance The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics.

Introduction
Burkholderia pseudomallei and Burkholderia mallei are closely related gram-negative bacteria that are the causative agents of melioidosis and glanders, respectively. These organisms are considered biothreat agents and are classified by the NIAID as class B pathogens. Using bacteriophage-mediated immunoscreening, Tiyawisutsri et al. identified four Trimeric Autotransporter Adhesins (TAAs) in B. mallei that were expressed during glanders infection [1]. TAAs are a family of outer membrane proteins that adhere to host cell surfaces, and thus have an important role in virulence of these pathogens [2]. Because TAAs are surface proteins with properties similar to hemagglutinins and invasins, these proteins are also referred to as Hep_Hag autotransporters or YadA-like autotransporters, in reference to the first known member of the family. Because of the differences in protein folds and binding partners between hemagglutinins and this family of proteins, we have adopted the more common and less confusing name of TAA [3]. All four TAAs identified by Tiyawisutsri et al. have homologs in B. pseudomallei. In total six TAAs were identified in B. mallei and nine in B. pseudomallei, indicating that these proteins are in relative abundance in comparison with other bacteria. TAAs have also been identified in Burkholderia cenocepacia, a respiratory pathogen associated with cystic fibrosis [4].
The earliest known and most well characterized TAA is YadA from Yersinia enterocolitica [5]. YadA is comprised of a C-terminal membrane anchored domain and an N-terminal domain, which is referred to as a passenger domain because it is believed to pass through the membrane anchored domain on its way outside the cell; hence, the term autotransporter. Based on the YadA primary structure, passenger domains are comprised of an N-terminal secretion sequence, a head region, and a stalk region. The head of YadA has been shown to bind collagen [6]. A crystal structure of the head domain of YadA contains a proposed collagen binding surface [7], and YadA appears to bind to the triple-helical structure of collagen without sequence specificity [8]. Although a structure of an entire TAA has not yet been solved, structures have also been determined for individual domains of other TAAs. These include several parts of the head domains of Hia from Haemophilus influenza [9,10] and BadA from Bartonella henselae [11]; the coiled-coil domains from YadA [12], Salmonella enterica [13], and UspA1 from Moraxella catarrhalis [14]; and the membrane anchoring domain of Hia [9,15]. A recent crystal structure of the entire esterase EstA non-trimeric autotransporter from Pseudomonas aeruginosa provides additional insights into autotransporter function [16].
In general, TAAs are highly variable in sequence and length, making them difficult to identify and define their domain boundaries [3]. The most highly conserved and identifiable region of a TAA is the membrane anchored C-terminal region, often referred to as the YadA domain. Outside of this domain, it is challenging to identify other whole domains using general domain approaches. In contrast, approaches that identify short sequence motifs or subdomains of up to 40 amino acids in length are considerably more successful [3]. These short sequence motifs include the N-terminal secretion sequence, neck regions which may contain up to 50% sequence identity [11], left handed parallel b-roll repeats (also referred to as Hep_Hag, YadA-like head or Ylhead repeats), and other elements not present in general domain databases. However, the regions surrounding these sequence motifs often contain low sequence similarity complicating the prediction of these elements and their boundaries. In one example, crystal structures of the Hia and BadA head domains reveal nearly identical folds for the Trp-ring and GIN domains despite no discernable sequence similarity a prior [11]. In another example, Hia contains three Trp-ring elements which as little as 18% sequence identity, but virtually super-imposable folds [9]. Despite low sequence similarity, these structures indicate the rules for the structural assembly of these domains. Several other short sequence motifs appear throughout TAAs that do not yet have known three dimensional structures. Structure elucidation of these motifs should aid in the development of new bioinformatic algorithms to identify these motifs in other TAAs.
In comparison with YadA, the nine TAAs from B. pseudomallei are considerably larger and more complex. Several B. pseudomallei TAAs contain multiple head regions, regions of low complexity, and are up to 2800 amino acids long [1]. To understand how B. pseudomallei binds to its host cell surface, we have investigated several B. pseudomallei TAAs using a structural genomics approach [17,18,19,20]. Here we present the crystal structure of a head domain from a B. pseudomallei trimeric autotransporter adhesin BpaA at 1.35 Å resolution. The BpaA head structure exhibits a novel fold of an intricately interwoven trimer that contains modular structural elements from other trimeric autotransporter proteins. Our work expands the foundation for understanding the structural basis for the adherence of infectious disease organisms to their hosts via TAAs.

BpaA Domain Architecture
The genome of B. pseudomallei contains a gene annotated as xadA or XadA-like protein based on sequence similarity to the TAA XadA from Xanthomonas oryzae [21]. To avoid confusion with the XadA protein from X. oryzae, we have adopted the term BpaA for the Burkholderia pseudomallei adhesion A protein investigated here. In B. pseudomallei, BpaA TAA is expressed as a 2757 amino acid long protein containing an N-terminal secretion peptide sequence, numerous head domains, a short coiled-coil domain, and a Cterminal membrane anchor domain ( Figure 1). Pfam [22] predicted the third head domain of BpaA to contain Hep_Hag and HIN2 domains (BPSS1434) [1]. To identify domain boundaries in the B. pseudomallei TAAs, the sequences were analyzed with InterPro [23] and the resulting Hep_Hag and HIN2 domains were aligned to the folded region of the YadA crystal structure [7] using the MUSCLE algorithm [24] iteratively, with re-alignment after each manual sequence extension or truncation. Ginzu [25] domain boundary predictions suggested that the full-length protein was all beta, even within the low-complexity segments. DISOPRED did not predict any disordered regions [26]. In addition, the entire BpaA sequence was entered into the domain annotation of trimeric autotransporter adhesins (daTAA) server [3] which predicted this region of BpaA to contain an FGG motif, a HANS motif, two Ylhead repeats, a HIN2 motif, a neck motif, and end with a coiled-coil ( Figure 1). With this combined information set, domain boundaries were manually selected to contain the aforementioned region resulting in a 178 amino acid long construct that spanned residues 2278-2455. Alignment of this sequence with the other three B. pseudomallei BpaA head regions (159-240, 39% sequence identity; 972-1148, 73%; and 2482-2653, 29%) matched reasonably well with the domain boundaries predicted by the daTAA server despite differences in the presence and number of TAA sequence motifs (119-240, 966-1143, and 2583-2645; Figure 1). Of those sequence motifs contained within this head region of BpaA, only the Ylhead and neck motifs have known structures from TAA homologs.

Crystallization and Structure Determination
Crystallization trials were attempted using a rational sparse matrix approach [27] with a construct containing residues 2278-2455 including a 21 amino acid long N-terminal affinity tag (see methods). Crystals were not observed for this construct, and therefore we tried in situ proteolysis using chymotrypsin to generate crystals [28]. Chymotrypsin cleaves after hydrophobic residues, which in this case is most likely after leucine 4 of the target protein sequence. Cleavage at this site would eliminate the 21 amino acid long affinity tag plus the first four amino acids of the target protein sequence. Although we do not know if in situ proteolysis was successful, crystals were not obtained in the absence of chymotrypsin. The crystals belonged to the rhombohedral space group H3 with unit cell dimensions (Table 1) consistent with one molecule in the asymmetric unit as predicted by the packing density [29,30]. A 1.35 Å resolution native data set was collected at the Advanced Light Source synchrotron (Table 1).
Attempts at molecular replacement using elements from YadA, Hia and Dex49a that have weak sequence similarity to BpaA as search models were unsuccessful. Therefore, we attempted de novo phasing. The third head region of BpaA does not contain methionine or cysteine residues, preventing structure determination via single or multiple wavelength anomalous dispersion methods (SAD/MAD) using selenomethionine or covalent heavy metal derivatization via mercury or platinum. Therefore, we attempted iodide ion soaking for SAD experimental phasing [31,32], which has proven successful for several other SSGCID targets (PDB entries 3K9G, 3KM3, 3KW3, 3LR0, 3LR5, 3LUV, 3MEN, 3MD7). A 2.05 Å resolution data set was obtained in house on a crystal soaked into a solution containing 1M potassium iodide (Table 1). Noting the twin fraction of 0.2, the number of iodide sites selected for phase determination was kept to a minimum to avoid including strong sites from the minor twin fraction. Four iodide sites were located for phase determination (see Methods). Each of these sites contained strong anomalous signal. Using the iodide/SAD experimental phases, the model was built initially using automated programs followed by manual model building. Of the 178 amino acid long construct, residues Ser5-Ala122 and Phe129-Asn177 were modeled. Although only 4 sites were used in determination of the experimental phases, a total of 9 iodide sites were located and built based on anomalous Fourier maps. All of the iodide ions bind to the surface of BpaA primarily at hydrophobic pockets, except for one iodide ion which binds along a 3-fold crystallographic axis within the coiled-coil at the C-terminus. The coiled-coil regions of other TAAs have been reported to bind ions [13]. The isomorphous native data set was refined directly against the protein only model from the iodidederived structure. Two additional residues could be built in the native data set including Ser123 and Ser178. Both structures are well refined with excellent geometry (Table 1) as determined by MolProbity [33].

BpaA head domain structure
The structure of the head domain of BpaA exhibits a tightly woven trimeric quaternary structure (Figure 2). No structural homolog could be found using the secondary structure matching (SSM) server [34] and thus, BpaA has a novel fold. The fold has less a/b character than most soluble proteins with numerous loop regions of undefined secondary structure. a1 stacks below a29 of an adjacent monomer, while a2 stacks on top of a199 from the third monomer of the trimeric complex ( Figure 2B and 2C). This combined a-helical stack is offset by 60u relative to a3 at the Cterminus ( Figure 2C and 2D). b1 stacks against a2 and a29 on the interior and b2 on the exterior. b3 follows a2 and forms the first b sheet in a stack with b49, b59, b69, and b7 on the interior of a left handed parallel b-roll [7]. The three copies of a1 (i.e. a1, a19, and a199) form a left handed heptad coiled-coil at the N-terminus of this third head domain of BpaA. However the sequence does not strictly follow the standard hxxhcxc pattern where h is a hydrophobic residue, c is a charged residue and x is another amino acid [35] since there are no charged residues in this stretch from Ile8 through Ser21.
Like other TAA head regions [11], the monomer is unlikely to be folded in the absence of the other two protomers of the trimer. Greater than half of the protein is buried by trimer formation given a solvent accessible surface area of 16,298 Å 2 and a buried surface area of 18,583 Å 2 . The interior of the protein along the 3-fold trimer axis is highly hydrophobic (Ile8, Ile11, Thr15, Gly19, Thr22, Leu26, Tyr44, Val59, Ile63, Ile66, Ile71, Phe74, Ile93, Val105, Ile107, Phe141, Val143, Ile152, Val155, Ala167, Gly170, and Leu173; Figure 3A). The exterior of the protein is hydrophilic ( Figure 3A) with a large acidic patch that spans the length of the protein, reflective of the predicted isoelectric point of 4.3 ( Figure 3B). The head domain structure of Hia (HiaBD1) also contains an acidic patch [9,10].
Bioinformatics approaches [3] predicted several sequence motifs in the B. pseudomallei BpaA third head domain (Figure 1) which were mapped onto the BpaA crystal structure (Figure 4). In this case, the sequence motifs predicted by the daTAA server overlap remarkably well with the sequence elements defined by the structure. One of these sequence motifs is the Ylhead (YadA-like head) left handed parallel b-roll, which was observed previously in the YadA head crystal structure [7]. Several sequence motifs or elements predicted from sequence and bioinformatics approaches [3] such as the FGG motif and the HANS motif were novel to the BpaA crystal structure (Figure 4). The structure explains why the few amino acids that define these motifs are highly conserved, whereas other residues within the motif are not well conserved. For example, the FGG motif contains cross-monomer stacked ahelices (a1 with a29) with a b-turn insert (b1-b2) ( Figure 4). FGG motifs typically contain a phenylalanine as the first residue, although BpaA contains the less common leucine residue at this position; twenty-seven of seventy-three sequences aligned in daTAA contain leucine [3]. The first residue (Leu26) must be highly hydrophobic to promote packing at the trimer interface and only appears as phenylalanine or leucine. Modeling a tryptophan or tyrosine residue at this site appears to induce a steric clash along the 3-fold trimeric axis, explaining why these residues are not observed at this position in this sequence motif. The next two residues (27)(28) can only be glycine, which would otherwise clash with the helical stacking of a1-a29 or with the packing of b2 with a2-a29. These residues are typically followed by the sequence gAxY (details are described on the daTAA server [3]). From this, Tyr32 stacks against a1 while forming a water-mediated interaction with Thr16. The end of a2 includes three additional modestly conserved residues. Ser58 is conserved as a Ser or Asn and induces a turn by forming a hydrogen bond with the backbone of Ala61. Val59 packs within the trimer interface. Gly60 is conserved as a small residue (Gly or Ala).
The HANS motif forms the first b-sheet in the stack of b3, b49, b59, b69, and b7 along the left handed parallel b-roll and forms four backbone hydrogen bonds with b49 ( Figure 4). The side chain of His75 forms hydrogen bonds with Asp82 and the backbone oxygen of Asn69. Ala76 resides in a small pocket created by the highly conserved Tyr7399 and Phe7499 that would not accommodate larger amino acids. The side chain of Asn77 forms hydrogen bonds with the backbone oxygen atoms of Thr86 and Gly87. Finally, Ser78 forms a hydrogen bond with the backbone nitrogen of Asp80, forming a turn that leads into the first Ylhead repeat. Thus, with the exception of the His75-Asp82 salt bridge, the HANS motif exclusively forms hydrogen bonding interactions with the backbone atoms of neighboring amino acids. Furthermore, the hydrogen bonding and b-sheet patterns explain why HANS motifs are only found preceding Ylhead repeats [3]. Finally, our structure contains the HIN2 region which has not previously been structurally characterized, although part of this region is disordered (residues 124-128). This region contains a b-sheet which stacks in left-handed parallel b-roll-like fashion on top of the previous Ylhead motif. Inserted within this region is a loop that packs against the neck motif, explaining why the HIN2 motif is only observed prior to the neck motif [3]. This motif is characterized by an FxG motif, two of which is observed back to back from Phe129 through Gly134 in BpaA. Both phenylalanine residues pack against a3, the coiled-coil that extends from the neck motif. We suspect that if our construct had a longer coiled-coil on the C-terminal end of the construct, the turn in the HIN2 domain would have been ordered.

Discussion
The third head domain of BpaA exhibits a highly interwoven trimeric fold in a manner reminiscent with other known TAA head domain structures YadA [7], Hia [9,10], and BadA [11]. These different TAAs achieve their tightly interwoven trimeric structures despite considerable sequence divergence and different domain architecture. For each TAA the monomer is composed of several sequence motifs ( Figure 5). In the case of BpaA, the Ylhead and neck motifs have been observed in other TAA structures ( Figure 5). The collagen binding Ylhead regions, with its characteristic left handed parallel b-roll and SVAIG-S sequence motif, align well between YadA and BpaA. This motif is present in the BadA head domain as well, although it was not part of the construct used for structure determination. In particular, the neck regions align well in structure and contain considerable sequence identity as previously analyzed [9,11]. The FGG, HANS and HIN2 motifs present in BpaA were not present in YadA, Hia or BadA. Our structure is in agreement with the bioinformatics of these modular elements and explains sequence and domain architecture conservation.
Like other TAAs, a considerable amount of the BpaA head domain does not have a-helix/b-sheet secondary structure. These regions prove difficult to identify by secondary structure prediction programs. As described previously, short sequence motif-based prediction methods fair better at predicting these motifs than general domain prediction methods [3]. The crystal structure of the third head domain from BpaA demonstrates the structural basis of three TAA sequence motifs of previously unknown structure. The structural basis demonstrated for the FGG, HANS and HIN2 sequence motifs by our BpaA structure should be useful for defining sequence restraints within structure prediction programs. In general, TAA sequence motifs are highly modular in nature and often rearranged in different TAAs, yet the structures of sequence motifs such as the Trp-ring motif remain highly similar despite considerable sequence divergence [9,11]. We predict that the same will hold true for the FGG, HANS, and HIN2 motifs described here once additional structures have been determined.
In comparison with the low sequence identity yet similar structural elements of other TAAs, BpaA may provide additional evidence for evolution of TAAs from a common ancestor [2,36]. We speculate that this may be common for other extracellular proteins or extracellular domains of membrane-anchored proteins. For example, periplasmic domains of two-component system histidine kinases have evolved to recognize a wide variety of different ligands, yet all exhibit a common PAS domain fold [37,38]. In addition, lipocalins are extracellular iron binding proteins that exhibit the same fold despite almost no sequence identity in an effort to evade host immune response [39]. Thus, TAAs from pathogenic organisms may exhibit similar general features to other extracellular proteins or extracellular domains of membrane-anchored proteins in terms of evolution and efforts to combat the immune response of their host.
Despite the fact that TAAs perform an important role in host cell infection, it appears that they have evolved with a high degree of sequence divergence and general architecture as a means to escape host immune response. Nevertheless, we note that several domains with highly different sequences exhibit the same fold across several TAAs from pathogenic organisms, perhaps due to a common ancestor. Therefore, antibodies or aptamers that target the global fold rather than the local structure of TAA head domains may hold potential as novel diagnostics.

Protein expression and purification
The third head domain of BpaA from B. pseudomallei strain 1710b (NCBI: YP 335617.1; gene BURPS1710B_A0459; UniProt Q3JLD6) spanning residues 2278 to 2455 was cloned into a pAVA0421 vector using ligation independent cloning (LIC) [40]. BpaA was expressed in E. coli using BL21(DE3)R3 Rosetta cells and autoinduction media [41] in a LEX Bioreactor. The frozen cells were resuspended in 200 ml of Lysis Buffer (20 mM HEPES, pH 7.3, 300 mM NaCl, 5% glycerol, 30 mM Imidazole, 0.5% CHAPS, 10 mM MgCl 2 , 3 mM b-mercaptoethanol, 25 units/ml of Benzo-naseH nuclease, and 0.05 mg/ml lysozyme). The resuspended cell pellet was disrupted on ice for 15 minutes with a Branson Digital Sonifier 450D (settings at 70% amplitude, with alternating cycles of five seconds of pulse-on and ten seconds of pulse-off). The cell debris was clarified by centrifugation on a Sorvall RC5 at 6,000 RPM for 60 min at 4uC. The protein was purified from the clarified cell lysate by immobilized metal affinity chromatography binding on Ni Sepharose High Performance resin (GE Biosciences, Piscataway, NJ) equilibrated with Binding Buffer (20 mM HEPES, pH 7.2-7.4, 300 mM NaCl, 5% glycerol, 30 mM Imidazole). The recombinant protein was eluted with 500 mM imidazole and was further resolved by size-exclusion gel chromatography (SEC, Superdex 75 26/60; GE Biosciences, Piscataway, NJ). Pure fractions collected in SEC Buffer (20 mM HEPES pH 7.0, 300 mM NaCl, 2 mM DTT, and 5% glycerol) as a single peak were pooled. The protein was concentrated, flash frozen, and stored in 280uC. The affinity tag was not removed prior to crystallization trials.

Crystallization
Crystals were grown using the sitting drop vapor diffusion method at 16 uC using either the JCSG+ or PACT sparse matrix screens from Emerald BioSystems [27]. For the native crystal, 0.4 mL of the protein stock solution at 24.9 mg/mL in 20 mM HEPES, 0.5 M NaCl, 5% Glycerol, 2 mM DTT (pH 7.0) with 0.1 mg/mL chymotrypsin was incubated with a similar volume of reservoir solution (10% PEG 1000 and 10% PEG 8000). The crystal was cryo-protected in the reservoir solution enhanced with 25% ethylene glycol. The crystal used for phase determination was grown against a reservoir of 0.  5.0.3 which has an ADSC Q315 detector. The data were reduced with HKL2000 [42] yielding a rhombohedral space group with one molecule in the asymmetric unit. Crystallographic statistics are presented in Table 1. A 2.05 Å resolution iodide data set was collected in house using a Rigaku Micromax 007-HF X-ray generator with Osmic VariMax HF optics and a Saturn 944 CCD detector. The data were reduced with XDS and XSCALE [43] yielding a data set in the rhombohedral space group isomorphous with the native crystal. The resolution of the iodide data set was limited by the size of the detector and the parameters used for data collection. Both the native and iodide crystals had a twin fraction of about 0.1-0.2 as determined by detwin analysis in the CCP4 suite [44].

Structure determination
The structure was solved by the single wavelength anomalous dispersion (SAD) method using iodide ions as the heavy atoms. Four iodide sites were located using Phenix [45]. Experimental phasing was performed using PHASER EP from the CCP4 suite [44]. Experimental electron density maps were density modified in PARROT. The initial model was built with BUCCANEER [46] and followed with additional building in ARP/wARP [47]. Additional iodide sites were identified using anomalous difference Fourier maps, and all iodide site occupancies were determined by refinement of the SAD data directly within REFMAC [48]. The final model was produced after numerous reiterative rounds of refinement in REFMAC and manual building in Coot [49]. In later rounds of refinement, the model was refined with amplitude based twin refinement. Refinement statistics are detailed in Table 1.


s2fieldsofstudy: Biology Medicine

text:
Differential HMG-CoA lyase expression in human tissues provides clues about 3-hydroxy-3-methylglutaric aciduria

3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients.

The disease usually appears in the first year of life after a fasting period or during intercurrent illness. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis, hypoketotic hypoglycaemia and a characteristic pattern of elevated urinary organic acid metabolites (Gibson et al. 1988;Schutgens et al. 1979;Wysocki and Hahnel 1986). Such organs as the liver and brain are frequently affected, and occasionally the pancreas and heart can also be involved (Gibson et al. 1994;Muroi et al. 2000;Urganci et al. 2001;Wilson et al. 1984;Zafeiriou et al. 2007;Zoghbi et al. 1986).
The pathophysiology of this disease is only partially understood. It may be explained by the deficit of an alternative energy source (ketone bodies), the intracellular accumulation of toxic organic acids or fatty acids or secondary carnitine deficiency (Kahler et al. 1994;Mitchell et al. 1995;). However, these interpretations were not based on a thorough understanding of the expression or activity of HL in the affected tissues. To our knowledge, HL has been examined only in liver, leukocytes and fibroblasts (Ashmarina et al. 1994;Wysocki and Hahnel 1976a;Wysocki and Hahnel 1976b) but not in other human tissues. This report is the first study of mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase in kidney, pancreas, testis, heart, skeletal muscle, and brain. The results may improve our understanding of the enzyme function and the involvement of these organs in 3-hydroxy-3-methylglutaric aciduria.

HL mRNA
HL mRNA was measured in Multiple Tissue cDNA (MTC) panels (Clontech) by a Real-Time quantitative PCR method using a TaqMan Gene Expression Assay (Assay HMGCL: Hs00609306_m1 Assay GAPDH:Hs99999905_m1) (Applied Biosystems). The levels of HL mRNA was calculated relative to the GAPDH control by the ΔΔCt method. The level of expression HL in skeletal muscle was arbitrarily taken as the calibrator to normalize the other tissues.

HL protein analysis
Human tissues: liver, kidney, pancreas, testis, heart, skeletal muscle, and brain were obtained within 10-12 h post mortem at autopsy from two male subjects at the Department of Pathology of our University. They were 65 and 70 years old, and representative samples of organs of approximately 1-2 g were taken from each subject. To minimize protein degradation, the bodies were kept at 4°C until autopsy and all the tissues were immediately shockfrozen in N2 and stored at −80°C until use.
To isolate the mitochondrial fraction from human tissues, we performed subcellular separation as described by Clinkenbeard et al. (1975). Mitochondrial proteins separated on a 15% SDS-PAGE were transferred to a 0.45-μM nitrocellulose membrane by transfer blot. The membranes were probed with a mouse monoclonal antibody against HL (Abnova) (1:1,000 dilution) and revealed with the kit SuperSignal West Dura Extended Duration substrate (Thermo Scientific) using a peroxidase-conjugated secondary anti-mouse antibody (Sigma, 1:1,000). The protein bands were quantified using Scion ImageJ v1.39u software (Scion) and normalized to micrograms of total protein obtained from 10 mg of tissue. The density of the band corresponding to the liver was taken as 100%.

HL activity
After subcellular separation, 20 μl of mitochondrial fraction of different tissues (about 200 μg of protein) was used to measure enzymatic activity. HMG-CoA lyase activity was assayed by the spectrophotometric assay described by Wanders et al. (1988), which measures the amounts of acetoacetate produced. The level of activity was expressed as mU/g wet weight tissue (one milliunit equals 1 nmol of substrate converted/min).

PAP measurements
Serum samples were collected in acute and inter-crisis periods from 15 patients with 3-hydroxy-3-methylglutaric aciduria and stored at −20°C. All patients were diagnosed biochemically, and most of them were also diagnosed genetically (Table 1). Human serum PAP (pancreatitis associated protein) concentration was determined in control and patients by a kit based on a sandwich immunoenzymatic system (PancrePAP, Dynabio).
The values of PAP in patients varied between 5.31-31.87 ng/mL (Table 1) within a normal range (<50 ng/mL) (Desjeux et al. 2002). Simultaneously, we also measured PAP protein in 11 normal subjects as controls, and no differences (range 6.12-32.61 ng/mL) were found.

Discussion
This is the first time that a complete study of mRNA, protein and enzymatic activity of HL has been reported in a broad range of human tissues. The findings on the expression level and enzymatic activity of HL in these tissues may help us to understand the pathophysiology of HL deficiency.
Our results show that the liver has the highest HL activity, followed by pancreas. Both organs showed high levels of mRNA, protein and HL enzyme activity. The liver is the organ most frequently affected in this deficiency, although involvement is usually mild, with elevated transaminases and hepatomegaly (Urganci et al. 2001;Wysocki and Hahnel 1986). Since the liver does not use ketone bodies as a source of energy (Robinson and Williamson 1980), its deficit is not the cause of the disease. However, ketogenesis is more active in the liver and the blockage of this pathway could result in an accumulation of toxic intermediate metabolites. Recently, Leipnitz et al. (2009) have demonstrated in vitro the oxidative stress caused in the liver by the accumulation of organic acids in this deficiency.
Pancreatitis is a potential complication in patients with organic acidemias, especially methylmalonic and isovaleric (Kahler et al. 1994) and less frequently in 3-hydroxy-3methylglutaric aciduria (Muroi et al. 2000;Wilson et al. 1984). The presence of HL protein in pancreas has been reported (MacDonald et al. 2007), but here we demonstrate that the pancreas has the highest enzymatic activity after the liver. This finding indicates that the pancreas has a high ketogenic capacity and suggests that, if ketone bodies regulate the release of insulin (Biden and Taylor 1983;Malaisse et al. 1990;Rhodes et al. 1985), some of them could be produced in the pancreas. In contrast, ketone bodies do not seem to play an important role as energy source in this organ. The finding of higher enzymatic activity in pancreas indicates that it may be more susceptible to toxic accumulation of metabolites. This hypothesis has been proposed to explain the pathogenesis of other organic acidemias of leucine catabolism (Gut et al.   Kahler et al. 1994). The possibility of a progressive deterioration of the pancreas, without evident clinical manifestations, led us to study the protein PAP in patients with 3-hydroxy-3-methylglutaric aciduria, in order to detect and prevent any possible pancreas damage. PAP is a more sensitive marker of pancreatitis than amylase, whose levels remain high even in absence of symptoms (Kemppainen et al. 1996). The serum studies from patients in different situations of crisis and between crises did not show pathologic elevations. However, the higher activity of HL enzyme and the severity of the complications support the need to monitor the pancreatic function in patients with HL deficiency. This study shows that, at different levels of mRNA, protein and enzymatic activity, HL is not found in the mitochondria from human brain. The absence of lyase activity in brain mitochondria is consistent with results obtained by McGarry and Foster (1969) and Bachhawat et al. (1955) in rat but it disagrees with the results of Shah (1982). Ketogenic activity by astrocytes has been described (Auestad et al. 1991;Guzmán and Blázquez 2004), but Auestad et al. (1991) have demonstrated that acetoacetate is mainly formed in astrocytes by deacylation of acetoacetyl-CoA and not by the action of the mitochondrial HMG-CoA cycle. In conclusion, despite the difficulties of studying the human brain immediately after death to avoid protein degradation, HL activity in mitochondria from adult human brain, if present, would be minimal. We suggest that the neurological alterations, frequently associated with this deficiency, are related to hypoglycaemia and to the absence of the only alternative substrate to glucose for the brain ketone bodies. Concomitantly, our results show that the organic acids would not be produced in situ, although they could cross the bloodbrain barrier of an immature brain (Wajner et al. 2004) and cause damage. Dilated cardiomyopathy has been described in two patients with 3-hydroxy-3-methylglutaric aciduria, one young male (Gibson et al. 1994) and one adult ). In this last case, the authors suggest that the cardiomyopathy results from impaired ketogenesis, intracellular fatty acid accumulation and a secondary carnitine deficiency. However, we found very little HL in heart tissue, from which we conclude that it does not regulate the entry of fatty acids to the heart and thus prevent their accumulation. Therefore, our results do not support the hypothesis of local accumulation of organic acids as a cause of the cardiomyopathy. In HL deficiency, heart disease could be caused by the lack of an alternative energy substrate. The heart is a continuously active muscle which uses various energy substrates depending on their availability (Kodde et al. 2007). Although ketone bodies are not an indispensable substrate, the added L-carnitine deficiency, which is caused by the HL deficiency, could alter the transport of fatty acids to the mitochondria for oxidation and the coupling between glycolysis and glucose oxidation (Allard et al. 2006). L-carnitine supplementation has been recommended for patients with organic acidurias to allow   (Chalmers et al. 1984), but it could also improve the heart function.
In HL deficiency, alterations in other organs such as kidney, testis and skeletal muscle have not been reported, although in our study the enzymatic activity in kidney was high or moderately high in testis and skeletal muscle. Surprisingly, in muscle, although the mRNA levels were very low, moderate HL activity was measured. Similar cases are reported in the literature (Lewin et al. 2001), which suggests that certain tissues may have a lower turnover of the HL protein versus an unstable mRNA. In testis, the low activity levels of HL compared with the high enzyme expression suggest that HL activity could be regulated after translation in this tissue. All these tissues use the ketone bodies as an alternative energy source, but they do not show strong dependence on them (Robinson and Williamson 1980). Moreover, they are less sensitive to the accumulation of toxic metabolites, in contrast to the brain (Wajner et al. 2004) or pancreas .
The results reported here help us to understand the enzyme function and pathophysiology of the 3-hydroxy-3methylglutaric aciduria. We suggest that pancreatitis should be included as a possible complication to seek in patients with HL deficiency, especially during acute episodes. Moreover, the addition of nutritional factors, such as L-carnitine, can be essential to prevent the development of cardiomyopathy.


text:
Tests of Integrin Transmembrane Domain Homo-oligomerization during Integrin Ligand Binding and Signaling*

Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for both inside-out and outside-in signaling, the role of TM homomeric association remains elusive. Formation of TM homo-oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo-oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo-oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, integrin TM homo-oligomerization is not required for integrin activation, ligand binding, or signaling.

Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic ␣ and ␤ domains associate in the resting state and separation of these domains is required for both inside-out and outside-in signaling, the role of TM homomeric association remains elusive. Formation of TM homo-oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo-oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo-oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, integrin TM homo-oligomerization is not required for integrin activation, ligand binding, or signaling.
Integrins are cell adhesion molecules that are essential for many biological functions such as cell migration, survival, and differentiation. These functions are accomplished by integrin bidirectional signaling across the cell membrane. Inside-out activation occurs when specific intracellular molecules such as talin and kindlin bind to the integrin cytoplasmic domain, leading to integrin conformational change and therefore high affinity for extracellular ligands. Then, binding of multimeric extracellular ligands results in outside-in signaling that is critical for many cellular processes. It has been shown that integrin bidirectional signal transduction requires integrin structural change and lateral distribution (clustering) (1,2).
Integrins are type I transmembrane (TM) 2 proteins consisting of two noncovalently associated ␣ and ␤ subunits, each with a large extracellular domain, a single-span TM domain, and a short cytoplasmic domain. Recent structural studies have greatly advanced our understanding of integrin conformational change during inside-out activation (3)(4)(5)(6)(7)(8)(9). Even though relatively short, the integrin TM/cytoplasmic domains play a crucial role in this process. It has been shown that the association of ␣ and ␤ subunit TM/cytoplasmic domains is critical for stabilizing integrins in the resting state (10 -16). When induced by binding of the ␤ subunit cytoplasmic domain through talin or other intracellular molecules, the TM/ cytoplasmic domains separate (17,18), driving integrin extension and shifting the ligand binding ␣/␤ headpiece to an open, high affinity conformation (7,19). Recently, the structure of the TM/cytoplasmic domains in the resting state was proposed based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface (16). In this structure, the ␣IIb GXXXG motif and its counterparts in the ␤3 TM domains associate with ridge-in-groove packing, and the ␣IIb GFFKR motif and the ␤3 Lys 716 in the cytoplasmic segments play a critical role in ␣/␤ association. The structures of complex and monomeric ␣ and ␤ subunit TM/cytoplasmic domains have also been solved by NMR (8,9,20,21). These studies have shed light on the structural basis of integrin TM/cytoplasmic domain signaling across the plasma membrane (22).
In contrast to the role of heterodimeric TM/cytoplasmic domain association and dissociation, that of homo-oligomerization of integrin TM domains in integrin signaling remains elusive. In 2001, an NMR study of this region by Li et al. failed to detect heterodimeric association between the ␣ and ␤ subunit TM/cytoplasmic domains using TM/cytoplasmic fragment peptides in micelles. Instead, they observed that the ␣ and ␤ fragments tend to form homodimers and homotrimers, respectively (23). Later, ␣IIb and ␤3 TM helices were confirmed to form homo-oligomers in bacterial membranes using the TOXCAT assay (24,25). The ␣ and ␤ homomeric interactions have been widely studied by computational modeling (26 -28). These studies showed that the homo-oligomerization interface and the heterodimerization motifs largely overlap, but it seems that homomeric interaction is less specific than the heterodimeric interaction (25). In 2003, asparagine mutagenesis in the TM region of ␤3 subunit (with most experiments on the mutation ␤3G708N) suggested that TM homo-oligomerization contributes to integrin activation and clustering (29). However, the mutation ␤3G708N, which was reported to enhance trimerization in detergent and increase ligand binding avidity in the transfected CHO cells (29), was later found to activate the integrin by changing ligand binding affinity rather than valence (13). Furthermore, mutations that disrupted homodimerization of integrin TM domains, which also disrupted heterodimerization because the two interfaces overlap, were shown to activate integrins for ligand binding, suggesting that TM domain separation is sufficient to activate integrins (14). Therefore, it was proposed that integrin TM homo-oligomerization is not a critical step for inside-out activation, but instead, it may help to stabilize the integrin in the high affinity state and be important for outside-in signaling (24).
It has been shown that integrin outside-in signaling requires both conformational change and clustering of integrins. However, the mechanism of how integrins transmit these signals across the plasma membrane through the TM/ cytoplasmic domains remains unknown. More specifically, it is unclear whether integrin TM homo-oligomerization plays any role in integrin clustering and signaling, even though it has been proposed that it provides structural basis for this process (29,30). Importantly, although ␣ and ␤ TM homooligomerization was found in micelles and bacterial cell membranes (14,23,25,29,31), it has never been observed in mammalian cell membrane using full-length integrins. On the contrary, the TM heterodimeric but not homomeric association of integrin fragments was detected using mini-integrin affinity capture method in mammalian cell membranes (32).
In this paper, we tested whether integrin TM domains form homo-oligomers in mammalian cell membranes using disulfide scanning of intact integrin ␣IIb␤3. Our results show that integrin TM domains do not form homo-oligomers before or after soluble ligand binding or during integrin inside-out and outside-in signaling.

EXPERIMENTAL PROCEDURES
Plasmid Construction and Transient Transfection-Plasmids coding for full-length human ␣IIb and ␤3 were subcloned into pEF/V5-HisA and pcDNA3.1/Myc-His(ϩ), respectively (33). Amino acid substitution in TM and TM proximal regions to cysteine was made using site-directed mutagenesis with the QuikChange kit (Stratagene). Constructs were transfected into 293T cells (American Type Culture Collection) using a FuGENE transfection kit (Roche Applied Science) according to the manufacturer's instructions.
Immunoprecipitation-Twenty-four hours after transfection, 293T cells were treated with 20 g/ml 2-bromopalmitate, metabolically labeled with [ 35 S]cysteine/methionine for 1.5 h before adding chase medium containing 500 g/ml cysteine and 100 g/ml methionine, and cells were cultured for 17 h overnight (12). Then, cells were detached and suspended in Tris-buffered saline (TBS) (10 6 cells in 100 l) supplemented with 5 mM Ca 2ϩ , 1 mM Mn 2ϩ with 3 mg/ml fibrinogen or 1 mM Mn 2ϩ with 50 M RGD peptide and incubated at room temperature for 30 min. Then, saponin was added to a final concentration of 0.02%, and 20 M CuSO 4 /100 M ophenanthroline was added by 10-fold dilution from stock solutions, and cells were incubated on ice for 10 min. Oxidation was quenched by adding an equal volume of TBS containing Ca 2ϩ and 5 mM N-ethylmaleimide. Cells were lysed by the addition of an equal volume of 2% Triton X-100 and 0.1% Nonidet P-40 in the same buffer for 10 min on ice. Cell lysate was centrifuged and immunoprecipitated with 1 g of anti-␤3 mAb AP3 and protein G-Sepharose at 4°C for 2 h. After three washes with lysis buffer, precipitated integrin was dissolved into 0.5% SDS sample buffer and subjected to nonreducing 7.5% SDS-PAGE and fluorography (12).
For ␣IIbW967C mutant, 2 mM DTT was added to the 35 Slabeled cells and incubated at 37°C for 10 min, washed twice with TBS, and then oxidized by Cu-phenanthroline on ice for 10 min. Oxidation was quenched by adding an equal volume of TBS containing Ca 2ϩ and 5 mM N-ethylmaleimide. Cells were lysed, and integrins were immunoprecipitated as described above.
To test whether integrins form TM homomeric disulfide after adhering to the immobilized fibrinogen, 35 S-labeled cells were seeded on the surface of 6-well plates precoated with immobilized fibrinogen and incubated at 37°C for 1 h. After washing twice with TBS, adherent cells were incubated on ice with Cu-phenanthroline and saponin and quenched with 5 mM N-ethylmaleimide. Cells were lysed, and integrins were immunoprecipitated as described above.
Ligand-induced Binding Site (LIBS) Epitope Expression-LIBS epitope expression was measured as described (34). Briefly, transfected cells suspended in HEPES-buffered saline supplemented with 5.5 mM glucose and 1% bovine serum albumin (BSA) were incubated with either 5 mM Ca 2ϩ , 3 mg/ml fibrinogen, or 50 M Gly-Arg-Gly-Asp-Ser-Pro peptide (GRGDSP) in the presence of 1 mM Mn 2ϩ at 37°C for 15 min, and then 10 g/ml anti-LIBS1 antibody was added. After incubation on ice for 30 min, cells were washed and stained with FITC-labeled anti-mouse IgG on ice for 30 min. The stained cells were subjected to flow cytometry, and LIBS epitope expression was expressed as the percentage of mean fluorescence intensity of anti-LIBS antibody relative to mean fluorescence intensity of the conformation-independent anti-␤3 mAb AP3.
Cell Spreading, Integrin Clustering, and Microscopy-Glass bottom 6-well plates (MatTek Corporation, Ashland, MA) were coated overnight at 4°C with 20 g/ml human fibrinogen in phosphate-buffered saline (PBS) at pH 7.4 followed by blocking with 1% BSA at room temperature for 1 h. The transiently transfected HEK293T cells were detached by trypsin/ EDTA and washed three times with DMEM. Cells were seeded on fibrinogen-coated dishes. After incubation at 37°C for 1 h, cells were washed 3 times with PBS and fixed with 3.7% formaldehyde in PBS at room temperature for 10 min or for integrin clustering, 10 g/ml anti-␤3 mAb AP3 was added at room temperature for 30 min followed by staining with 10 g/ml FITC-conjugated goat anti-mouse IgG for 30 min at room temperature before fixation. 400 nM cytochalasin D was added as control before seeding.
Differential interference contrast imaging was conducted on a Leica TCS SP2 spectral confocal system, coupled to a DM IRE2 inverted microscope with 63ϫ oil objective. For the quantification of cell spreading area, outlines of 100 randomly selected adherent cells were generated, and the number of pixels contained within each of these regions was measured using ImageJ software (National Institutes of Health, Bethesda, MD).
Cell Adhesion-Cell adhesion on immobilized human fibrinogen was assessed by the measurement of cellular lactate dehydrogenase activity as described (1). Briefly, cells suspended in HEPES-buffered saline supplemented with 5.5 mM glucose, 1% bovine serum albumin, and 1 mm Ca 2ϩ were added into flat bottom 12-well plates (1 ϫ 10 5 cells/well) that had been precoated with 20 g/ml fibrinogen and blocked with 1% BSA. After incubation at 37°C for 1 h, wells were washed three times with HEPES-buffered saline supplemented as indicated above. Remaining adherent cells were lysed with 1% Triton X-100, and lactate dehydrogenase activity was assayed using the Cytotoxicity Detection Kit (lactate dehydrogenase) (Roche Applied Science) according to the manufacturer's instructions. Cell adhesion was expressed as a percentage of bound cells relative to total input cells.

RESULTS
Integrin TM Domains Do Not Form Homo-oligomers before or after Soluble Ligand Binding-Previously, cysteine mutagenesis and heterodimeric disulfide scanning were used to identify the integrin ␣IIb and ␤3 TM interface in mammalian cell membrane successfully (12,16). Here, we used singlecysteine mutations of the ␣IIb and ␤3 TM region (␣IIb residues 965-995 and ␤3 residues 691-723; Fig. 1A) and applied a similar method to determine whether the ␣IIb or ␤3 TM domains form homo-oligomers during integrin signaling. As suggested by the TOXCAT assay (24,25) and predicted by computer modeling (26 -28), the ␣IIb TM helix forms homodimers, and the ␤3 TM helix forms homotrimers, using surfaces that are similar to those in the heterodimeric interface. If this homomeric interface is actually formed in the mammalian cell membrane, we expected that some singlecysteine mutations of these residues would form homodimeric disulfide bonds in the presence of an oxidation catalyst such as Cu-phenanthroline. In addition, 2-bromopalmitate was used to block cysteine palmitoylation, which can inhibit disulfide formation; and saponin was used to increase the permeability of Cu-phenanthroline and the efficiency of disulfide bond formation as described previously (16). To confirm the efficiency of disulfide bond formation in the presence of Cu-phenanthroline, several cysteine pairs (one from ␣ and the other from ␤) at the TM or TM proximal region, which have been shown to form heterodimeric disulfide bonds (12,16), were used as controls.
As shown in Fig. 1, the cysteine pair ␣971C/␤697C formed a heterodimeric disulfide bond in the presence of Cu-phenanthroline (Fig. 1B), as described previously (12). By contrast, none of the single-cysteine mutants of ␣IIb TM regions formed homomeric disulfide bonds under these conditions except ␣IIbW967C (Figs. 1B and 2). As reported previously (12,13), ␣IIbW967C, when co-transfected with wild-type ␤3, formed a homomeric disulfide bond in the resting state, and therefore, the ␣IIb␤3 integrin associated to form a disulfidebridged tetramer (␣IIbW967C/␤3) 2 . This residue is located outward, away from the heterodimeric interface or predicted homodimeric interface (12,13). When DTT was added, the disulfide bond was reduced. After DTT was removed and Cuphenanthroline was added to catalyze disulfide formation, only a trace amount of homomeric disulfide was formed on the cell surface ( Fig. 2A). We further traced the disulfide bond formation of ␣IIbW967C mutant after 35 S labeling by lysing cells and immunoprecipitating the protein at different time points (Fig. 2B). After 30 min of labeling, ␣IIb precursor was formed. Then after 1 h, mature ␣IIb subunit was formed, and a trace amount of disulfide-bonded ␣IIb was observed. In 1.5 h, a significant amount of disulfide-bonded dimeric ␣IIbW967C was formed. This suggests that the homomeric disulfide bond is formed during biosynthesis and post-transla-tional modification. Because this disulfide bond of the ␣IIb mutant is formed during biosynthesis and in the resting state in which heterodimeric TM association is not affected, we excluded this mutant for the following studies. None of the other 30 ␣IIb cysteine mutants formed a homomeric disulfide bond (Fig. 1B and supplemental Fig. 1), and neither did any of the 34 ␤3 cysteine mutants ( Fig. 1C and supplemental  Fig. 2). These results suggest that the ␣IIb and ␤3 TM helices do not form homomeric associations when ␣IIb␤3 is in the resting state.
To determine whether the ␣ and ␤ TM domains form homo-oligomers after soluble ligand binding, we used Mn 2ϩ to activate integrin ␣IIb␤3 followed by binding of soluble fibrinogen and ligand-mimetic RGD peptide. It has been shown that ligand binding induces integrin conformational changes that expose the LIBS epitopes, which are located at the interfaces between the headpiece and tailpiece and between the ␣ leg and ␤ leg so that they are buried in the bent conformation but exposed in the extended conformation (35,36). We used anti-␤3 LIBS mAb LIBS1 (37) to determine the conformational states of the ␣IIb␤3 in the presence of Mn 2ϩ with fibrinogen or RGD. The LIBS1 bound poorly to wild-type ␣IIb␤3 in Ca 2ϩ (Fig. 1D), suggesting that it is in the bent conformation. The binding significantly increased when fibrinogen with Mn 2ϩ or RGD with Mn 2ϩ was added (Fig. 1D), suggesting that these ligands bound to integrins, and they stabilized integrins in the more extended conformation. Disulfide scanning was used to test whether ␣ and ␤ TM domains form homomeric interface after integrin bound to soluble fibrinogen and RGD peptide under the same condition (Fig. 1, B and C, and supplemental Figs. 1 and 2), and no homomeric disulfide formation was observed for any mutants except the ␣W967C as described above, suggesting that after ligand binding, even though integrins are stabilized in the more extended state and probably with two separating legs and TM/cytoplasmic tails, homo-oligomerization does not occur for TM/cytoplasmic tails in mammalian cell membrane.
Integrin TM Domains Do Not Form Homo-oligomers during Inside-out Signaling-We designed experiments to test whether the integrin TM region forms homo-oligomers during inside-out activation. It is well known that the GFFKR motif in the ␣IIb cytoplasmic domain is crucial for maintaining integrin in the resting state. When the two phenylalanines of the GFFKR motif are mutated to alanines (GAAKR mutant), integrins are activated to bind ligands with high affinity. Thus, this GAAKR mutant can be used to mimic integrin inside-out activation (12). We confirmed that the mutant bound fibrinogen constitutively; in addition, it bound LIBS1 much better than the wild-type in the Ca 2ϩ condition (Fig. 1D), indicating that the mutation shifts integrin toward more extended conformation, well mimicking inside-out signaling. We made six single-cysteine mutants using this GAAKR construct and co-transfected them with the wild-type ␤3 integrin. None of the ␣IIb integrin cysteine mutants formed a homomeric disulfide bond (Fig. 3). Similar results were obtained when 34 ␤3 single-cysteine mutants were co-transfected with the ␣IIb GAAKR mutant; none of these mutants formed a homomeric disulfide bond (Fig. 3). These results strongly sug- gest that during integrin inside-out activation across the mammalian cell membrane, TM homomeric association does not occur. Therefore, the TM homo-oligomerization does not play any role in this process.
Integrin TM Domains Do Not Form Homo-oligomers after Adhering to Immobilized Fibrinogen-After binding to immobilized ligands, integrins on the mammalian cell surface will transmit outside-in signals. We carried out a series of experiments to address whether TM homomers form during this process. We randomly chose two ␣IIb and two ␤3 cysteine mutants and used them for cell adhesion and cell spreading assay. HEK293T cells transiently transfected with wild-type and mutant ␣IIb␤3 were seeded on fibrinogen-precoated dish surfaces at 37°C for 1 h. The amount of adherent cells was assessed by quantifying the cellular lactate dehydrogenase activity. All selected mutants could adhere to the immobilized fibrinogen similarly to the wild-type receptor, whereas the disulfide-bounded mutant (␣968C/␤693C) showed much less adhesion, and DTT treatment which was used to disrupt the disulfide bond was able to recover its adhesion to the similar level to the wild-type with DTT (Fig. 4A). To test whether the single-cysteine mutations could affect cell spreading, HEK293T transient transfectants were coated on the immobilized fibrinogen at 37°C for 1 h, followed by fixation and microscopic analysis. Cells transfected with all single-cysteine mutants demonstrated cell spreading to a level similar to those transfected with the wild-type receptor. By contrast, the disulfide-bounded mutant (␣968C/␤693C) showed no spreading, and DTT treatment restored cell spreading (Fig. 4, B and  C). These results confirmed that dissociation of the TM heterodimers is required for integrin outside-in signaling as described previously using CHO cell transfectants (1). We also chose four cysteine mutants (␣G972C, ␣G976C, ␤L705C, and ␤L712C), which were proposed to be critical for the homomeric interactions (25,31), and used them for the cell spreading assay. Cells transfected with all mutants showed cell spreading similar to those transfected with the wild-type integrin (data not shown). Taken together, our results indicate that the single-cysteine mutations in this region do not affect the integrin adhesion to immobilized fibrinogen, nor do they affect outside-in signaling.
It is widely believed that lateral association (i.e. "clustering") of integrin heterodimers, which occurs as a consequence of multivalent ligand binding (38,39), plays a major role in outside-in signaling (40 -42) (for review, see Ref. 43). It was also shown that ligand binding can directly lead to and stabilize separation of integrin cytoplasmic domains (44), and this integrin conformational change is critical for outside-in signaling as well (1). However, it remains unclear whether the integrin TM homomeric interaction after the TM helix separation is critical for integrin clustering. To assess formation of integrin clustering, HEK293T transfectants were allowed to adhere to fibrinogen-coated substrates followed by fixation and staining with fluorescent anti-␣IIb␤3 antibodies. Under these conditions, wild-type integrins and all cysteine mutants could be readily detected in clustered patterns (Fig. 5). Interestingly, although the disulfide-bounded ␣968C/␤693C mutant is defective for cell spreading, it formed similar cell clusters as others, in contrast to the negative control in which the actin filament disruptor cytochalasin D prevented integrin clustering (Figs. 4 and 5).
We then further determined, under the same conditions, whether integrin TM domains formed homo-oligomers in the plasma membrane. HEK293T cells transfected with a variety of ␣IIb and ␤3 cysteine mutants were seeded on a fibrinogen substrate at 37°C for 1 h. After cells fully adhered and spread on immobilized fibrinogen, Cu-phenanthroline was added to catalyze disulfide bond formation. Four cysteine mutation pairs, ␣968C/␤693C, ␣971C/␤697C, ␣972C/␤697C, and ␣955C/␤723C, were used as controls to confirm the efficiency FIGURE 3. Integrin TM domains do not form homo-oligomers during inside-out activation. Samples were processed as in Fig. 1 and subjected to nonreducing 7.5% SDS-PAGE and fluorography. A, cysteine mutations of the ␣IIb GAAKR mutant were co-transfected with wild-type ␤3 integrin. No homomeric disulfide bond was formed for any of the ␣IIb cysteine mutants. B, cysteine mutations of the ␤3 TM region were co-transfected with the ␣IIb GAAKR mutant. No homomeric disulfide bond was formed for any of the ␤3 cysteine mutants.  Fig. 3). Because these integrins adhered to the immobilized fibrinogen, clustered on the cell surface and transmitted outside-in signaling leading to cell spreading, the results suggest that homomeric association of the integrin TM domains is not important for integrin functions. Similar results were obtained when CHO cells were used (data not shown), suggesting that integrin TM/cytoplasmic domains do not form homomeric interaction during integrin outside-in signaling in mammalian cells.

DISCUSSION
It has been shown that TM heterodimeric association stabilizes integrins in the resting state; when integrins are activated by intracellular signals, two TM/cytoplasmic tails separate, leading to conformational change of the ligand binding extracellular regions, resulting in high affinity for ligands (5, 8, 9, 12, 13, 16 -18, 45). Therefore, equilibrium between the dissociated monomers and associated heterodimers of the TM domains is critical for integrin inside-out activation. Based on the observation that integrin TM helices formed homo-oligomers using recombinant peptides in detergent (23,29,31), the GALLEX assay in bacteria (45), the TOXCAT assay in bacteria (24,25), and the computational modeling (26 -28), TM homomeric association has been suggested to be important for integrin activation (14,29,31). Mutagenesis studies on the proposed TM homomeric interface residues showed that these mutations could lead to integrin activation, suggesting that heterodimer dissociation is sufficient to activate integrins (14). Therefore, a push-pull model was proposed in which after dissociation of the TM heterodimer, integrins are stabilized by TM homomeric association (14).
Here, our experiments on mammalian cell transfectants using full-length integrin ␣IIb␤3 showed that the ␣IIb and ␤3 TM domains do not form any homomeric association in the resting state, nor do they form after soluble ligand binding. The only mutant that formed a homomeric disulfide is ␣IIbW967C, as reported previously (12,13). We found that this homomeric disulfide was formed during biosynthesis and in the integrin resting state, and dissociation of the TM heterodimer is not required for its formation. Therefore, we do not think that the same homomeric association occurs during integrin inside-out activation or outside-in signaling. We found that except for this mutant, none of the other 30 ␣IIb and 34 ␤3 cysteine mutants formed a homomeric disulfide bond before or after binding to soluble ligands. In addition, during integrin inside-out activation, the integrin TM domains do not form any homomeric association that can be detected by disulfide scanning. Integrins must be regulated to be activated and deactivated quickly in mammalian cells, even within seconds. It has been shown that detachment of integrin from ligands in the trailing edge is critical for integrin functions (46), and mutations that constitutively active integrins result in malfunctions (47)(48)(49)(50). Therefore, it is unlikely that integrin TM domains form more stable homomeric association during inside-out signaling.
We further showed that TM homomers do not form upon to binding to ligands on a substrate and subsequent integrin clustering. Although integrin clustering is known to participate in integrin signaling pathways (40,(51)(52)(53), the exact mechanisms of integrin clustering remain elusive. One mechanism proposes that the integrin TM homo-oligomerization promotes integrin clustering (29). Studies have established that extracellular ligand binding triggers integrin conformational changes that promote oligomer assembly through TM fragments (54 -56). In this study, we observed that after adhering to immobilized fibrinogen integrins clustered on the cell surface, but we did not detect any integrin TM homomeric interaction under this condition. Interestingly, the heterodimeric disulfide-bonded integrin formed clusters, suggesting that even the dissociation of integrin ␣/␤ TM domains is not required for integrin clustering. In addition, no evidence has been shown that the integrin extracellular domains form homomeric interaction, and therefore, it is unlikely that they induce integrin clustering during signaling. Cells expressing ␣IIb␤3 wild type and mutants were seeded on fibrinogen-coated surface for 1 h at 37°C in the presence or absence of cytochalasin D (Cyto D). Attached cells were then stained with anti-␤3 mAb AP3 in the presence or absence of cytochalasin D for 30 min at room temperature, followed by staining with FITC-conjugated goat antimouse IgG for 30 min at room temperature. After fixation, cells were subjected to confocal microscopy. FIGURE 6. Integrin TM domains do not form homo-oligomers after cells adhering to the immobilized fibrinogen. Samples were processed as in Fig. 1 and subjected to nonreducing 7.5% SDS-PAGE and fluorography. A, none of the ␣IIb cysteine mutants formed a homomeric disulfide bond after adhering to the immobilized fibrinogen. B, none of the ␤3 cysteine mutants formed a homomeric disulfide bond after adhering to the immobilized fibrinogen.
Taken together, our study suggests that integrin clustering is likely induced by bringing several integrins physically close together by multimeric ligands, and the TM/cytoplasmic domain homomeric association is not required for this process. Additional evidence is needed to define the mechanisms of integrin clustering further.
Even though the TM separation is not required for integrin clustering, it is critical for integrin outside-in signaling (1). We showed that all cysteine mutants could adhere and spread on the fibrinogen-coated surface, suggesting that they could transmit signals into the cells after binding to immobilized ligands. However, we did not observe any TM homomeric disulfide formation in the same condition, indicating that the TM homomeric association does not occur during integrin outside-in signaling. Based on the previous FRET studies (44), we assume that ligand-induced TM separation is likely coupled to cytoplasmic domain separation, implying that ␣-␤ cytoplasmic domain interactions somehow constrain or inhibit kinase activation (1,13,17,44,57). One possibility is that separation of TM and cytoplasmic domains induced by multimeric ligand binding is required for kinases or other intracellular proteins to bind integrin cytoplasmic tails. On the other hand, some studies have shown that certain tyrosine kinases, such as Src, associate constitutively with integrin ␣IIb␤3 in platelets, and platelet adhesion to fibrinogen causes a rapid increase in Src activation (58). Thus, another possibility is that the association of the integrin TM and cytoplasmic domains somehow constrains the activity of integrin tailbound kinases, whereas dissociation of the TM and cytoplasmic domains releases these constraints. In either case, binding of multimeric ligands brings integrins together and at the same time, induces dissociation of the TM/cytoplasmic domains, resulting in kinase autophosphorylation and activation. The current study strongly suggests that during this process, the TM homomeric association does not play any role in kinase activation.
In conclusion, our study shows that the integrin TM homomeric association does not occur in mammalian cell membranes before or after soluble ligand binding, during insideout activation or outside-in signaling. Therefore, conformational change induced by intracellular signals or extracellular ligands, more specifically, separation of TM and cytoplasmic domains, but not the homomeric association, is critical for integrin bidirectional signaling.