Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM – a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site prediction tools. In the independent testing, the high sensitivity and specificity of the proposed method demonstrate the predictive effectiveness of the identified substrate motifs and the importance of investigating potential kinases for viral protein phosphorylation sites. Viruses are biological agents that interrupt and manipulate normal cellular functions Protein phosphorylation is the most widespread and well-studied post-translational modification (PTM) in eukaryotic cells Further understanding of viral protein phosphorylation is essential due to its importance with regard to viral progression. However, there is a great deal of difficulty in experimentally identifying viral protein phosphorylation sites using mass spectrometry-based techniques; thus, computational methods for identifying protein phosphorylation sites have been proposed. Existing phosphorylation site prediction tools can be classified into three categories: general or non-specific, organism-specific, and kinase-specific In phosphorylation, it is known that substrates are targeted by kinases according to a specific pattern. Specific amino acid residues at certain positions of a protein greatly affect the specificity of a particular kinase Based on the current state of research, there is still a lack of understanding as to what kind of host kinases specifically phosphorylates viral proteins. Therefore, we are motivated to develop a method to investigate the substrate motifs and identify potential host kinases for viral protein phosphorylation sites. The identification of kinases is deemed important as these are heavily pursued pharmaceutical targets due to their mechanism role in various diseases K-means clustering method K which denotes the number of samples to be obtained from the negative set is defined by the number of corresponding positive data. This resulted in an equal number of positive and negative S, T, and Y fragments respectively in the three data sets as shown in In order to investigate the residues surrounding the phosphorylation sites, sequence fragments are extracted using a window size of 11 centered on S, T, and Y. A window size of 11 consists of 11 amino acid residues placed from position −5 to 5. Fragments having a phosphorylated residue on position 0 are obtained and regarded as positive data while fragments centered on non-phosphorylated residues are regarded as negative data. As shown in It is observed that the phosphorylated sequences in each subgroup clustered using maximal dependence decomposition (MDD) show a conserved motif representing its substrate site specificity. The flanking amino acids (−5 ∼ +5) of the non-redundant phosphorylation sites, which are centered on position 0, are graphically visualized as sequence logos using WebLogo. Maximal dependence decomposition is executed multiple times with varying values in order to obtain the most optimal minimum cluster size. Setting the minimum cluster size to 50 for pSer data yielded 7 clusters as shown in For virus pThr and pTyr data, the minimum cluster size is set to ten. Similar to the process of selecting the minimum cluster size for pSer, increasing the minimum cluster size did not result in any clusters and further lowering of the minimum cluster size resulted in several similar clusters. This resulted in three clusters in pThr as shown in In order to identify potential host kinases for human virus substrates, the motif of each MDD-generated viral protein phosphorylation cluster is compared with the discovered human kinase substrate specificities. As shown in Further analyzing the matched motifs, a literature survey is done in order to find studies that experimentally identify human kinases which phosphorylate specific virus protein substrates. Previous studies To demonstrate the effectiveness of MDD clustering method, the MDD-detected motifs are compared with two well-known motif discover tools, Motif-X The cross-validation process includes the selection of the threshold parameter for each model. The threshold parameter is a specific bit score that serves as the cutoff value of HMMsearch for determining matching query sequences for an HMM According to a five-fold cross-validation evaluation, the predictive performance of MDD-clustered HMM performs significantly better than non-MDD clustered HMM of pSer, and pThr. As shown in An independent test is done due to the possibility of an over-fit of the models in the training set which may lead to an overestimation of its predictive performance In order to further evaluate our approach, each predicted phosphorylation site resulting from the independent test is studied. A survey on existing literature is done by referencing UniProt To further demonstrate the effectiveness of the proposed method, the independent testing data is used to make a comparison between the performances of three popular kinase-specific phosphorylation site prediction tools, Predikin 2.0 In this study, viral protein phosphorylation sites found in humans are further elucidated by means of identifying their potential catalytic human kinase. The study is done using experimentally verified viral protein phosphorylation sites obtained from virPTM In addition to the consideration of linear sequence motifs, substrate recruitment is very important in the investigation of kinase substrate specificity. However, with limited information regarding kinase-specific phosphorylation sites on viral proteins, the substrate recruitment of kinases could not be investigated for the viral protein phosphorylation data. This is the main reason why this work develops a computational method to investigate potential kinase substrate motifs for viral protein phosphorylation sequences. The approach offers the scientific community clues regarding human kinases that may be responsible for the phosphorylation of human virus proteins. It is important to note, however, that the further acquisition of experimentally verified viral protein phosphorylation sites is required to identify more meaningful viral protein phosphorylation motifs. Also, a more abundant set of experimentally verified kinase-annotated human phosphorylation sites could be used to improve the collection of substrate motifs. These developments could benefit our method by allowing the identification of more potential human kinases catalyzing virus proteins.K-means clustering method K-means clustering is performed based on sequence identity. The value of K which denotes the number of samples to be obtained from the negative set is defined by the number of corresponding positive data.In this work, the experimentally verified data of viral protein phosphorylation sites are collected from virPTM Xmn represents the number of sequences that have the amino acids of group m in position Ai and have the amino acids of group n in position Aj, for each pair  with i≠j. Emn is calculated as mRX  =  m1X+ …+m5X, CnX  =  1nX+ …+5nX, and X denotes the total number of sequences. If a strong dependence is detected  between two positions, then the process is continued as described by Burge and Karlin The phosphorylated fragments from the obtained training set are used to investigate the motif signatures of phosphorylated virus proteins. In order to explore the conserved motifs from a large data set, MDD is applied to cluster all phosphorylated fragments into subgroups that show statistically significant motifs. MDD is a methodology that groups a set of aligned signal sequences to moderate a large group into subgroups that capture the most significant dependencies between positions. Previous studies n+1 that is centered on phosphorylation sites. Next, a positional weighted matrix (PWM) (2n+1)×m elements is used to represent each MDD-cluster or kinase group, where 2n+1 stands for the window length and m consists of 21 elements for the 20 types of amino acids and for one terminal signal. Then, the Euclidean distance Meanwhile, in order to identify the various human kinase substrate specificities, human phosphorylated proteins annotated with their catalytic kinases are collected from Phospho.ELM. The phosphorylation sites are extracted using a window size of 11 and are grouped together according to its annotated human kinase. Each human kinase group is then graphically visualized as sequence logos using WebLogo. The motifs of the MDD-generated viral protein phosphorylation clusters and the visualized substrate specificity of human kinases are compared. A substrate-kinase match is selected by comparing the conservation of amino acids in each position (−5 ∼ +5) appearing as obvious motifs in the visualized sequence logos of each virus MDD clusters and human kinase. Fragments of amino acids are extracted from MDD clusters and human kinase groups using a window length of 2In this work, profile HMM is built from the site sequences of each MDD-clustered subgroup. An HMM describes a probability distribution over a potentially infinite number of sequences For each model of the MDD-clustered subgroups, a threshold parameter is selected as a cut-off value in identifying potential positive data from a query Prior to the construction of a final model, the predictive performance of the models with varying parameters are evaluated by performing k-fold cross validation. In doing k-fold cross validation, the training data is divided into k groups by splitting each dataset into approximately equal sized subgroups. In one round of cross-validation, a subgroup is regarded as the test set, and the remaining k-1 subgroups are regarded as the training set. The cross-validation process is repeated k rounds, with each of the k subgroups used as the test set in turn. Then, the k results are combined to produce a single estimation. The advantage of k-fold cross-validation is that all original data are regarded as both training set and test set, and each data is used for testing exactly once Figure S1Distribution of the collected viral protein phosphorylation data.(TIF)Click here for additional data file.Figure S2The analytical flowchart of MDD.(TIF)Click here for additional data file.Table S1Statistics of experimentally verified phosphorylation sites from virPTM, UniProtKB, and Phospho.ELM.(DOC)Click here for additional data file.Table S2pSer Virus MDD-clustered Motifs.(DOC)Click here for additional data file.Table S3Refined pSer Virus MDD-clustered Motifs.(DOC)Click here for additional data file.Table S4pThr Virus MDD-clustered Motifs.(DOCX)Click here for additional data file.Table S5pTyr Virus MDD-clustered Motifs.(DOCX)Click here for additional data file.Table S6Comparison of pSer and pThr motifs between MDD clustering and Motif-X.(DOCX)Click here for additional data file.Table S7Comparison of pSer and pThr motifs between MDD clustering and MoDL.(DOCX)Click here for additional data file.Table S8The amino acids group used in MDD clustering.(DOCX)Click here for additional data file.Table S9Summary of Human Viruses.(DOCX)Click here for additional data file.Table S10Summary of Human Kinases.(DOCX)Click here for additional data file.


Xanthomonas resistance 21, flagellin sensitive 2, and elongation factor-Tu receptor, possess intrinsic protein kinase activity and regulate downstream signaling through phosphorylation events. Here, we focus on the phosphorylation events of plant PRRs that play important roles in the immune response. We also discuss the role of phosphorylation in regulating mitogen-associated protein kinase cascades and transcription factors in plant immune signaling.Plants are continuously challenged by pathogens including viruses, bacteria, and fungi. The plant immune system recognizes invading pathogens and responds by activating an immune response. These responses occur rapidly and often involve post-translational modifications (PTMs) within the proteome. Protein phosphorylation is a common and intensively studied form of these PTMs and regulates many plant processes including plant growth, development, and immunity. Most well-characterized pattern recognition receptors (PRRs), including  Arabidopsis and 84.8, 12.3, and 2.9% in rice  that affect their conformation, activity, stability, and localization. These PTMs, which are often reversible, are highly specific regulators of many cellular processes . Current in rice . Phospho in rice  will notArabidopsis, more than 1,170 phosphopeptides from 472 phosphoproteins were identified after treatments with flg22 or xylanase, both of which elicit immune responses in Arabidopsis cell cultures , recognizes a conserved sulfated peptide called AxYS22, derived from the Xanthomonas oryzae pv. oryzae (Xoo) protein Ax21  and elongation factor (EF)-Tu receptor (EFR), which recognize the flg22 peptide from flagellated bacteria and the EF-Tu-derived peptide elf18, respectively -associated kinase 1 (BAK1), an RD kinase, was initially identified as a positive regulator of brassinosteroid responses. BAK1 forms an in vivo ligand-dependent complex with the BRI1 receptor , or glycine (G) before the conserved catalytic aspartate (D) residue. All plant receptor kinases (RKs) characterized to date that carry the non-RD kinase motif are involved in recognition of conserved microbial signatures . In contreceptor . Furtherreceptor . BAK1 nureceptor . The ricDe novo phosphorylation of a FLS2/BAK1 complex is clearly detectable in cells 15 s after the addition of flg22 using in vivo labeling with short pulses of [33P]orthophosphate . Targeted mutagenesis of the XA21 JM domain indicated that amino acids Ser686, Thr688, and Ser689 are autophosphorylated and required to maintain XA21 protein stability . For example, the protein phosphatase 2C XB15 no longer interacts with XA21S697A, indicating that Ser697 in the JM domain is critical for interaction with XB15 .In animals, signal transduction is often regulated by phosphorylation of residues in the JM domain of RKs . It is ntability . Transgetability . Thr705  of XA21 . The XA2ith XB15 . AutophoArabidopsis, the FLS2 JM residue Thr867 appears to be analogous to Thr705 in XA21  also abolished flg22-induced generation of ROS . Initially, MEKK1 activates MPK4 which was previously shown to negatively regulate the defense response ] families . In animand rice . TherefoArabidopsis, revealed that many WRKYs are directly regulated by MAPKs (Arabidopsis WRKY33 (AtWRKY33) is induced by conserved microbial signatures, such as the oomycete-derived peptide Pep25 (in vivo in response to Botrytis cinerea infection and by MPK4 at least in vitro (PDF1.2 (plant defensin 1.2).High-density protein microarrays, employed to identify downstream factors of MAPKs in by MAPKs . For exade Pep25 . Subsequin vitro . Phosphoin vitro . Perceptin vivo complex with MPK4 and MKS1  in yeast, suggesting that these WRKYs regulate gene expression downstream of MPK4 . It was and MKS1 . Howeverand MKS1 . This suand MKS1 . Followiin vitro, which binds to the W-box element in the OsPR1 gene promoter .To date, there are only a few reports suggesting that MAPKs phosphorylate rice transcription factors in response to pathogen infection. For example, OsMPK17-1 phosphorylates OsWRKY33 promoter . OsMPK17in vitro . TransieOsPR1 and OsPR10 (Xoo (Although a role for MAPK-mediated phosphorylation of WRKYs has not been demonstrated for XA21-mediated immunity, several WRKYs interact directly with XA21 in yeast. For example, OsWRKY62, identified as XB10 in a yeast two-hybrid screen using the XA21 intracellular domain as bait, interacts with the XA21 kinase domain in rice protoplasts  and negad OsPR10 . AdditioR10 (Xoo . Althougin vivo PRR phosphosites. This lack of success may be due to the observed rapid endocytosis and/or degradation of PRRs following perception of conserved microbial signatures (in vivo genetic studies.Recognition of conserved microbial signatures by PRRs is critical to plant survival. PRR activation induces rapid autophosphorylation, leading to phosphorylation of many other proteins. Despite the importance of phosphorylation in PRR-mediated immunity, only a few phosphorylation sites of PRRs have been identified. Those phosphorylation sites were initially found by targeted mutagenesis. Although recent advances in phosphoproteomic analyses using mass spectrometry have greatly expanded our capability to identify phosphopeptides , this apgnatures , which lArabidopsis cells successfully revealed several differentially phosphorylated proteins such as auxin efflux carriers and respiratory burst oxidase protein D (In addition to slow progress in identifying residues phosphorylated on the PRR itself, other proteins that could serve as targets of PRR phosphorylation have not yet been identified. Therefore, there is still a gap in our understanding of how precisely PRRs are able to initiate early signaling events such as activation of MAPKs, a rapid calcium influx and an oxidative burst. To answer these fundamental questions, it will be essential to identify such target proteins and to determine how these proteins regulate downstream events. Phosphoproteomic comparison is one method that can identify proteins that become phosphorylated during PRR-mediated immunity. For example, quantitative phosphoproteomic analyses performed on flg22- or xylanase-treated rotein D .Another important goal is to identify the substrates of MAPKs that are phosphorylated during PRR-mediated immunity. To date, only a few transcription factors have been shown to be phosphorylated by MAPKs during PRR-mediated immune responses. Studies utilizing protein microarrays, protein complex immunoprecipitations, and phosphoproteomic analyses will continue to uncover additional transcription factors and other potential MAPK targets, further contributing to our understanding of the role of phosphorylation in plant immune responses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: DDR1 is a receptor tyrosine kinase that signals in response to collagen.Results: Mutagenesis at the 211NDS glycosylation site enhances receptor dimerization and results in ligand-independent receptor autophosphorylation.Conclusion:N-Glycosylation of DDR1 plays a critical role in maintenance of the inactive state of the receptor dimers.Significance: These studies highlight a new structural feature that regulates DDR1 activation. N-glycosylation at the Asn211 residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved 211NDS N-glycosylation motif, but not other N-glycosylation sites , result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr484, Tyr520, Tyr792, and Tyr797. The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved 211NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that  A) , human embryonic kidney epithelial HEK-293 cells (CRL-1573), and breast cancer HCC1806 cells (CRL-2335) were all obtained from the American Type Culture Collection . COS1 and HEK-293 cells were cultured in Dulbecco's modified Eagle's medium, and HCC1806 cells were cultured in RPMI 1640 medium. All media were supplemented with 10% fetal bovine serum (FBS), 2 m513 of human DDR1b (Ab92564) (here referred to as Tyr(P)513) was purchased from Abcam . The specificity of this antibody for phosphorylated DDR1b, and not for DDR1a, was confirmed in our laboratory (data not shown). Transferrin receptor (TfR) mAb  was from BD Transduction Laboratories . A mAb against Myc was a generous gift from Dr. Guri Tzivion . Rat tail collagen type I  was purchased from Trevigen .A rabbit polyclonal antibody (pAb) against the C-terminal region of DDR1 (sc-532) and an antibody against GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology Inc. . Goat pAb against the N-terminal region of DDR1 (AF2396) was from R&D Systems . An anti-phosphotyrosine monoclonal antibody (mAb), clone 4G10®, referred to as anti-Tyr(P) (4G10), was from EMD Millipore . A phospho-specific rabbit pAb that recognizes the phosphorylated TyrHomo sapiens), NP_054699.2; chimpanzee (Pan troglodytes), NP_001038967.1; monkey (Macaca mulatta), AFH28699.1; orangutan (Pongo abelii), XP_002816688.1; cow (Bos taurus), NP_001069480.2; boar, (Sus scrofa), NP_001116577.1; panda (Ailuropoda melanoleuca), XP_002928892.1; dog (Canis lupus familiaris), XP_003639473.1; mouse (Mus musculus), NP_031610.1; rat (Rattus norvegicus), NP_001159494.1; frog (Xenopus laevis), NP_001083540.1; and zebra fish: (Danio rerio), XP_001345829.4. Sequence alignment was generated using ClustalW2. The prediction of N-linked and O-linked glycosylation sites was performed using NetNGlyc 1.0 Server and NetOGlyc 4.0 Server, respectively.The amino acid sequences of DDR1 used here were as follows: human  and the human IgG2 Fc  cDNAs as templates. The resultant fragments were then cloned into the pcDNA3.1/Myc-His(−)-A expression vector. Amino acid substitutions (N211Q and R105A) in the DDR1b ECD-IgG2 Fc construct were made by site-directed mutagenesis, as described above. The sequences of all wild-type and mutant constructs were verified by DNA sequencing. Expression of the recombinant proteins was accomplished by transient transfection, essentially as we previously described  were cloned into the expression vector pcDNA3.1/Myc-His(−)-A (Invitrogen) using conventional cloning approaches. Human DDR1a and DDR1b each harboring a Myc tag at the C-terminal end were constructed as described previously . DDR1a ant sites  were allescribed .m acetic acid) was then added to the serum-free media for 2 h at 37 °C, and then the cell lysates were obtained as described below. In the washout experiments, the collagen-containing media were aspirated after the 2-h incubation time, and the cells were washed (four times) with warm phosphate-buffered saline (PBS) followed by addition of fresh warm serum-free media. At various times (1–24 h), the cells were lysed for analyses. To obtain the cell lysates, the cells were washed twice with cold PBS and then lysed in RIPA buffer  supplemented with protease inhibitors , 10 mm NaF, and 1 mm sodium orthovanadate. The cell lysates were cleared by centrifugation at 13,000 × g at 4 °C for 10 min; protein concentration was determined using the BCA kit (Thermo Fisher Scientific Inc.), and the lysates were frozen at −80 °C until used.Cells were split the day before transfection to 60–70% confluence in 60-mm dishes. The next day, equal amounts of the appropriate expression vectors were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Four hours after transfection, the medium was aspirated, and the cells were washed twice with PBS, followed by incubation  in serum-free medium before stimulation. Rat tail collagen type I (10 μg/ml) or vehicle control (20 mm CaCl2 and 1 mm MgCl2) and then biotinylated with 0.5 mg/ml EZ-link-sulfo-NHS-biotin (Thermo Fisher Scientific Inc.) for 30 min. For control, a parallel plate of cells received PBS/CM without biotin . Then the biotin solution was removed, and cells were washed with PBS/CM, and the reaction was stopped by the addition of 50 mm NH4Cl for 10 min on ice. The cells were then lysed with RIPA buffer and centrifuged at 13,000 × g for 10 min. Equal protein amounts of lysates (400 μg) were then mixed with 120 μl (50% slurry) streptavidin-agarose resin (Thermo Fisher Scientific Inc.) and incubated overnight at 4 °C to capture biotinylated proteins. The mixtures were then briefly centrifuged, and the bound (beads) and unbound (supernatants) fractions were separated and collected. The beads were washed four times with cell harvest buffer . The bound biotinylated proteins were then eluted with 2× reducing Laemmli SDS-PAGE sample buffer and boiled. The bound, unbound, and total lysate fractions (40 μg each) were resolved by reducing 8% SDS-PAGE followed by transfer to a nitrocellulose membrane. The blots were probed with anti-Tyr(P) (4G10) antibodies for detection of phosphorylated receptor, and with anti-Myc antibodies for detection of total receptor. The blots were also probed with antibodies to the TfR and GAPDH to evaluate cell-surface proteins and cytosolic proteins level, respectively.COS1 cells seeded in 60-mm tissue culture dishes were transfected with wild-type or mutant DDR1 constructs, as described . After s513 antibody to DDR1b (Ab92564), and the other blot was probed with anti-Myc antibody. The latter blot was also reprobed with an antibody against GAPDH without stripping.Lysates of stimulated and unstimulated cells transfected with DDR1 cDNAs were divided into two fractions, and equal amounts of protein from each treatment were resolved by reducing 8% SDS-PAGE followed by immunoblot analyses into two duplicate blots. One blot was probed with anti-Tyr(P) mAb (clone 4G10®) or with anti-Tyr(P)2 glass coverslips were rinsed with PBS, and nonspecific sites were blocked with 0.1% BSA in PBS . Cells were then incubated (1 h) on ice with 5 μg/ml of the pAb to DDR1 AF2396 diluted in PBS supplemented with 0.1% bovine serum albumin (BSA). After washing with PBS, the cells were fixed with 2% paraformaldehyde in PBS for 15 min at room temperature and washed with PBS supplemented with 0.75% w/v glycine. The cells were then incubated  with a 1:500 dilution of FITC-conjugated donkey anti-goat IgG antibody (Jackson ImmunoResearch) and a 1:5000 dilution of DAPI (Invitrogen) in PBS + 0.1% BSA. Following repeated washes with PBS, the coverslips were mounted on slides with anti-fade reagent (Invitrogen). The samples were examined and photographed using a ×60 oil-immersion objective with a Leica TCS SP5 laser scanning confocal microscope at the Microscopy, Imaging, and Cytometry Resources Core of Wayne State University, School of Medicine. For flow cytometry, COS1 cells transfected with empty vector or vectors containing the WT or the N211Q DDR1b cDNA were harvested using an enzyme-free cell dissociation solution (Millipore) 48 h post-transfection. After washing once with PBS + 1% FBS (wash buffer), the cells were incubated with either purified goat serum or a goat anti-human DDR1 pAb  for 1 h on ice. Following two washes with wash buffer, the cells were incubated (45 min on ice) with 10 μg/ml donkey anti-goat IgG conjugated with Alexa 488 (Invitrogen). The cells were then washed twice with wash buffer and analyzed for green fluorescence by flow cytometry using an LSR II Analyzer (BD Biosciences). DAPI was added to the cells prior to flow cytometry to discriminate between live and dead cells.COS1 cells grown on 22-mmg, and the supernatants (containing the recombinant proteins) were saved for affinity purification using HiTrap Protein G HP Columns , according to the manufacturer's instructions. After extensive washes with PBS, the Fc-tagged proteins were eluted with 0.2 m glycine HCl buffer, pH 2.5, followed by neutralization with 1 m Tris-HCl buffer, pH 9.0. Purified proteins were concentrated by a 50-kDa cutoff spin filter (Millipore) and dialyzed against PBS using Slide-A-Lyzer Dialysis Cassettes, 20K MWCO (Thermo Fisher Scientific Inc.). The purity of the recombinant proteins was determined by Coomassie Blue staining after reducing 7.5% SDS-PAGE analysis. Protein concentration of the purified proteins was determined by measuring absorbance at 280 nm and calculated by using the molecular weight of 69,395.53 g/mol  and an extinction coefficient of 119,480 cm−1m−1.COS1 cells were seeded in 100-mm dishes transiently transfected with the WT, N211Q, and R105A Fc-tagged DDR1b ECD cDNAs and cultured for 2 days in serum-free media (7 ml/dish). Two days later, the media were collected, and cell debris was pelleted at 3,000 × m acetic acid before adding to the flat-bottom 96-well plates for an overnight incubation at room temperature. Blocking buffer (0.05 mg/ml κ-casein in PBS supplemented with 0.05% Tween 20) was then added to the wells and the plates were incubated for 1 h at room temperature. Then the blocking buffer was aspirated, and each well was washed once with blocking buffer. The DDR1b ECD-Fc proteins were diluted in blocking buffer at the indicated concentrations and then added to the wells in triplicate. The plates were incubated at room temperature for 3 h, and then the wells were washed with blocking buffer. Goat anti-human IgG Fc antibody conjugated with horseradish peroxidase (Millipore) (1:5000 dilution in blocking buffer) was then added to each well and incubated for 1 h at room temperature. After a final wash, the bound recombinant fusion proteins were detected by adding to each well Substrate Solution  followed by Stop Solution (2 n H2SO4) . The absorbance in each well was measured using a microplate reader set to 450 nm, with a wavelength correction set to 540 nm. The data were fit with GraphPad Prism 5  using a three-parameter, one-site specific binding model with shared top and bottom values.The relative affinity of the recombinant DDR1 ECD-Fc proteins to immobilized collagen I was determined by ELISA, essentially as described previously , but witm acetic acid, as a negative control (unstimulated cells). The cells were then lysed with 20 mm HEPES buffer, pH 7.5, containing 90 mm NaCl, 50 mm NaF, 5 mm Na4P2O7, 1% Triton X-100, 10 mm NaF, and 1 mm sodium orthovanadate, supplemented with protease inhibitors . Followed by a brief centrifugation, protein concentration was determined, and aliquots of the lysates were supplemented without or with the amine-to-amine cross-linker BS3 (Thermo Fisher Scientific Inc.)  and incubated for 30 min at room temperature. The reaction was quenched with 40 mm Tris, pH 7.5, for 15 min. Then, control and cross-linked lysates (500 μg each) were subjected to an immunoprecipitation procedure by incubating (1 h on ice) the lysates with 0.5 μg/ml anti-DDR1 pAb sc-532. The mixtures were then supplemented with protein A-agarose beads (Thermo Fisher Scientific Inc.) and incubated overnight at 4 °C. The beads were then washed four times with HEPES buffer, and the captured immune complexes were released with 2× reducing Laemmli SDS sample buffer. After a brief centrifugation, the supernatants were boiled and resolved by 6% reducing SDS-PAGE, followed by immunoblot analyses using the goat anti-DDR1 antibody AF2396. The blots were also probed with anti-Tyr(P) mAb 4G10® to detect phosphorylated receptors. The same cross-linking procedure was used to identify endogenous DDR1 complexes in breast cancer HCC1806 cells without and with collagen I stimulation.Cross-linking of DDR1 in cell lysates was performed essentially as described . Brieflym glutathione in 75 mm NaCl, 10 mm EDTA, pH 7.5, for 15 min per treatment at 4 °C with gentle agitation. After addition of the reducing solution, the cells were washed twice with cold PBS/CM and lysed with RIPA buffer as described above. A plate of biotinylated cells was maintained at 4 °C for 30 min, as a control of total surface DDR1. These plates labeled “TS” did not receive reducing solution; instead, they received PBS/CM. As a negative control, at time 0 min, a plate was treated with glutathione directly after the biotin labeling. The lysates were centrifuged , and the supernatants were collected. Protein concentration was then determined in each supernatant using the BCA method. An equal amount of protein from each lysate was then incubated (overnight at 4 °C) with 50 μl of immobilized streptavidin-agarose resin (Thermo Fisher Scientific Inc.) to pull down biotinylated proteins. The beads were washed four times with harvest buffer , and the bound biotinylated proteins were eluted with 2× reducing Laemmli SDS sample buffer, boiled, and resolved by 8%, reducing SDS-PAGE followed by immunoblot analyses. The total and endocytosed DDR1 was detected with anti-Myc antibodies. The blots were then reprobed with a mAb to human TfR. Densitometric analysis of the bands of internalized DDR1 in the same blot was performed with the ImageJ program . The density (area per square unit) was plotted against time and expressed in arbitrary units. The relative amount of internalized DDR1b proteins at each time point was calculated as the percentage of biotinylated proteins from the total surface levels of DDR1b (100%).This assay was used to follow the internalization of WT and N211Q DDR1b in unstimulated and stimulated cells. Briefly, serum-starved COS1 cells transfected to express WT or N211Q DDR1b were treated without or with 10 μg/ml collagen I for 30 min at 37 °C. The cells were then washed with PBS/CM and immediately surface-biotinylated on ice with the cleavable sulfo-NHS-SS-biotin reagent for 30 min. Upon completion of the biotin labeling of the surface receptor population and subsequent blocking, warm serum-free media (37 °C) were added to the dishes, which were returned to the 37 °C incubator. At the indicated times (0–40 min), the respective dishes were removed from the incubator and cooled down in ice to halt internalization, and the media were aspirated and the cells washed with cold PBS/CM. Cell surface-bound biotinylating reagent was stripped by incubating the cells twice with 50 mm urea. The lysates were then subjected to reduction, alkylation, and trypsin digestion, as described  overnight at room temperature. Peptides were desalted on a C18 Sep-Pak Plus cartridge (Waters Associates), eluted with 25% acetonitrile, and lyophilized to dryness. Phosphotyrosine-containing peptides were immunoprecipitated from 1.8 mg of lyophilized lysate using 10 μg of pY100 antibody  and 30 μl of protein G beads (Thermo Fisher Scientific Inc.). Immunoprecipitated peptides were eluted in 40 μl of elution buffer , and the beads were removed by centrifugation at 5,000 × g for 3 min. Eluted peptides were then transferred to a fresh tube, and 2.5 μl of a heavy peptide standard mix was added per sample to allow for normalization of precipitated endogenous peptide levels between runs. Heavy peptides sequences and amounts are detailed in the supplemental Table 1. Samples were analyzed using a Q-Trap 4000 (ABSciex) mass spectrometer using a previously published procedure ) as follows: 10 min from 0 to 10% B, 45 min from 10 to 34% B, 10 min from 34 to 47% B, and 10 min from 47 to 100% B. Transitions were monitored for endogenous and heavy phosphopeptides as detailed in supplemental Table 1. Peak areas were calculated using Analyst version 1.5 software, and peak areas for endogenous peptide transitions were normalized to the corresponding transitions for the spiked heavy peptide standards. Data from the mass spectrometry analyses were expressed relative to total receptor levels, as measured by immunoblot densitometry.HEK-293 cells were transiently transfected with WT and N211Q DDR1b cDNA using the calcium phosphate method, as described previously . Fifty hescribed , 19. Bri4AG4)  reveals that the structures are highly similar. As a result, we were able to model the triple-helical collagen strands that were shown bound to the structure of DDR2 into the structure of DDR1, which allowed the visualization of collagen binding with respect to the domain and the surface of the membrane. The kinase domain was constructed using its x-ray coordinates (PDB code 4BKJ). For the transmembrane segment, we used secondary structure prediction servers PSIPRED  . The str PSIPRED  to prediN-glycosylation sites in DDR1 reveal two sites located within the DS-like domain at residues 211 and 260 and two sites within the EJXM region at residues 371 and 394 (A and B). Crystallographic studies of the DS-like domain of human DDR1 confirmed the presence of N-glycosylation at both the Asn211 and Asn260 positions  .Analyses of the  and 394 , A and Bositions . Howeverexamined B 20), s, sN-glycvolution C.N- and O-glycosylation sites , regardless of collagen exposure. In contrast, treatment with collagen I induced a strong DDR1b phosphorylation in COS1 cells expressing WT DDR1b , which migrates as a species of ∼120–125 kDa. In the absence of collagen, no WT receptor phosphorylation was detected . Like WT receptor, the mutants N260Q, N371Q, and N394Q were all phosphorylated in response to collagen I albeit at lower levels , whereas none of these mutants were activated in the absence of collagen I . Interestingly, we found that a pool of the N211Q DDR1b mutants displayed receptor phosphorylation regardless of collagen I presence . Moreover, collagen addition did not alter significantly the levels of N211Q constitutive phosphorylation . Similar results were observed with a DDR1a isoform harboring the N211Q substitution (data not shown) and expression of the N211Q mutant in HEK-293 cells (data not shown). Analyses of total cell lysates using anti-Myc antibodies showed that all the DDR1 proteins were detected as doublets, which represent precursor and mature receptor forms . We also noticed that, with the exception of N371Q , all of the mutants displayed a slight reduction in electrophoretic mobility in reducing SDS gels when compared with WT DDR1b , which is consistent with the disruption of N-linked glycosylation in the DDR1b mutants. The exception in the case of N371Q may stem from the possibility that the 371NSS motif actually is not occupied with glycans , and therefore they were not pursued in this study.To assess the relative contribution of the glycosylation sites in DDR1, we generated receptor mutants by site-directed mutagenesis of the  glycans , or alte211NDS site is important for regulation of DDR1 phosphorylation, and that our observation with a glutamine substitution at the Asn211 position is not restricted to the specific nature of glutamine, we generated two additional mutants, N211A and S213A, at the N-glycosylation motif 211NDS , we generated a double DDR1b mutant, in which both the Asn211 and Lys655 residues were substituted for Glu and Ala, respectively, to generate an N-glycosylation and a kinase-dead double mutant (N211Q/K655A). The Lys655 residue is located within the KD of DDR1, and it is critical for receptor autophosphorylation, when exposed to collagen I  . This result indicates that the collagen I-independent activation of the N211Q mutant is mediated by receptor autophosphorylation.To further verify that the f 211NDS . As showllagen I A, lane 4 lane 4) . Using t lane 4) A, lane 5Asupplemental Fig. 3 and supplemental Table 1). These sites include the highly conserved activation loop phosphorylation sites Tyr792 and Tyr796/797 as well as IJXM sites Tyr484 and Tyr520. All four sites that were measured using the SRM-MS assay showed similar fold change increases in phosphorylation versus the wild-type receptor suggesting that there is no selective site-specific preference for tyrosine phosphorylation in the N211Q mutant. Note that detection of phosphorylation in WT DDR1b in the absence of collagen by the SRM-MS assay represents background phosphorylation, which is below the detection limit of the immunoblotting technique. In addition, using an antibody recognizing Tyr(P)513 . Collectively, these data suggest a key role for N-glycosylation at the conserved 211NDS site in regulation of the activation state of DDR1.To characterize the specific tyrosine residues on the N211Q mutant that are phosphorylated in the absence of collagen I, we employed SRM-MS to quantitatively compare the phosphorylation states of WT and N211Q DDR1b in HEK-293 cells. We have previously employed a similar approach to map the tyrosine phosphorylation profile of DDR2 . These ayr(P)513 , 27, a tA, lanes 1 and 2). In contrast, N211Q was activated regardless of collagen I stimulation . Upon withdrawal of collagen, WT DDR1b maintained detectable phosphorylation up to 1 h, after which time receptor activation was no longer detected under these conditions. The N211Q mutant was activated in the absence of collagen , and addition of collagen did not significantly alter receptor phosphorylation . Interestingly, in contrast to WT DDR1b, the phosphorylated state of the N211Q mutant was readily detected even after 24 h of collagen withdrawal. Taken together, these studies suggest that glycosylation at the Asn211 site of DDR1 regulates the temporal dynamics of receptor inactivation, even in the absence of ligand. Moreover, these results also show that ligand withdrawal inactivates the phosphorylation state of WT DDR1 with kinetics inconsistent with sustained activation.Previous studies demonstrated that DDRs, unlike other RTKs, require relatively long incubations times ∼15–90 min) with collagen to accomplish maximal receptor activation in adherent cells 0 min wit, 29. MorFig. 4A, stimulation with collagen I resulted in WT DDR1b phosphorylation and distribution of the activated receptor at the cell surface (avidin-bound) and the intracellular compartment (unbound fraction) . Intracellular phosphorylated WT DDR1b represents endocytosed receptor . In contrast to WT DDR1b, the N211Q DDR1b mutant displayed phosphorylation in the absence of collagen stimulation, and the autoactivated mutant receptor was readily detected in the avidin-bound fraction (cell-surface proteins) , indicating that only the N211Q mutant is phosphorylated at the cell surface in the absence of collagen. Moreover, similar levels of WT and N211Q proteins were detected at the cell surface in the absence of collagen . Activated N211Q, but not WT receptor, was also detected in the intracellular fraction in unstimulated cells . This indicates that active N211Q DDR1b is also located intracellularly.The current model of DDR1 activation postulates that DDR1 traffics to the cell surface where it binds to collagen, leading to receptor autophosphorylation . HoweverFig. 5A, lanes 7–9) (representing input prior to avidin fractionation) with anti-Tyr(P) mAb 4G10® revealed activated receptors only in lysates derived from stimulated cells expressing WT receptor  and unstimulated cells expressing N211Q DDR1b , as expected. Analyses of the surface, intracellular, and lysate fractions for total receptor expression with the anti-Myc antibody revealed comparable expression levels of WT and mutant DDR1b . As a control for the subcellular pulldown, the samples were also probed with an antibody to the TfR, a known surface protein, which was readily detected in the avidin-bound fraction , as expected, and in the cell lysates . The presence of the N211Q mutant at the cell surface was also detected by immunostaining of nonpermeabilized COS1 cells and by flow cytometry using specific DDR1 antibodies . Collectively, these data show that in the absence of collagen stimulation, activated N211Q mutant is detected both intracellularly and at the cell surface. Moreover, the intracellular localization of activated N211Q suggests that the collagen-independent autophosphorylation of N211Q occurs during receptor trafficking to the cell surface.Analyses of the total cell lysates , consistent with receptor endocytosis . In contrast, the N211Q mutant displayed a reduced rate of receptor internalization, which was not altered in the presence of collagen I. Taken together, these data suggest a role for N-glycosylation at the Asn211 site in regulation of DDR1 internalization. Moreover, the deficient internalization of the N211Q mutant further supports the hypothesis that the intracellular pool of the autophosphorylated mutant  is due to autoactivation during trafficking to the cell surface.Previous studies demonstrated that DDR1 is rapidly internalized after collagen I stimulation . In agreFig. 5ocytosis . Interesocytosis , 31. At Asupplemental Fig. 5), as reported previously (A). Because of the presence of the human dimerizing Fc tag, the purified ECDs are able to generate stable SDS-resistant disulfide-linked homodimers, which under nonreducing conditions display a relative mass of ∼180 kDa . Binding assays of the ECD homodimers to immobilized collagen I revealed a dose-dependent and saturable binding of WT and N211Q proteins to collagen I with similar affinities (DK = ∼3–5 nm). In contrast, the R105A mutation abrogated collagen binding, as expected . Taken together, these results suggest that glycosylation at the Asn211 site of DDR1 does not significantly contribute to collagen binding, and therefore the inability of the N211Q mutant to undergo collagen I-induced endocytosis cannot be attributed to reduced collagen binding.Because the N211Q mutant did not respond to collagen I-induced activation and endocytosis, we asked whether this behavior was due to the inability of the mutant receptor to bind collagen I. Therefore, we measured the relative binding affinities of WT and N211Q DDR1b proteins to collagen I using a solid-phase binding assay. To accomplish this task, we expressed the ECDs of these proteins fused to the human IgG2 Fc fragment at the C-terminal end, as described , 23, 32.eviously . The Fc-eviously A. Becausexpected , demonstN-glycosylation in regulation of receptor dimerization (3 (A) and DDR1 dimerization was reported to occur during receptor trafficking . These species represent the monomeric forms under the denaturing and reducing conditions of SDS-PAGE, consistent with the noncovalent nature of DDR1 dimers . These forms are likely to represent dimers and monomers of WT and N211Q DDR1b. Interestingly, relatively higher levels of dimers and reduced levels of monomers were detected in cross-linked N211Q-containing extracts when compared with lysates containing WT receptors , suggesting increased ligand-independent dimerization in the N211Q mutant. Moreover, only the monomeric (noncross-linked) and dimeric (cross-linked) forms of N211Q, but not of WT DDR1b, were phosphorylated , as revealed with the anti-Tyr(P) antibody (4G10). These results indicate that the N211Q dimers are also activated in the absence of ligand. Taken together, these findings suggest a critical role for N-glycosylation at the Asn211 residue in regulation of DDR1 dimerization.Previous studies with the epidermal growth factor receptor (EGFR) showed a key role for rization , 34. Theation  to untreated HCC1806 lysates led to the appearance of a DDR1 immunoreactive form of >220 kDa and reduction in the levels of DDR1 monomers , consistent with presence of receptor monomers and dimers in unstimulated cells, in agreement with previous studies , suggesting that collagen stimulation promotes receptor dimerization. Analyses of receptor phosphorylation demonstrated that both the monomeric and dimeric forms of DDR1 were activated only in the presence of collagen, as expected . Hence, collagen stimulation is associated with both increased receptor dimerization and activation.The detection of cross-linked dimeric DDR1 forms in the COS1 system prompted us to examine the state of DDR1 dimerization in a natural cellular system. HCC1806 is a breast cancer cell line that expresses high levels of endogenous DDR1 . Therefo studies , 11. ColN-linked glycans at the 211NDS sequon in regulation of the autoinhibitory state, dimerization, and internalization of DDR1. The widespread conservation of the 211NDS sequon within the DS-like domain of DDR1 throughout evolution and the fact that only alterations at this site disrupted kinase auto-inhibition are consistent with this notion and in agreement with multiple evidence demonstrating a key role for N-glycosylation in regulation of RTKs  that control the autoinhibitory state of DDR dimers are still unknown. The data presented in this report point to a critical role for  of RTKs –38. Thusfunction , 39. In proteins , 41. Intreceptor .et al. (A). Collagen-binding site is >80 Å distant from the KD (A and B). Transduction of signal would take place via a conformational change produced by the altered dynamics as a result of the first collagen binding event  that translate into downstream conformations that counteract the propensity of the transmembrane domain helices of DDR1 to generate active dimers (211, the structural transition or dynamic modification would mimic that of the triple-helical collagen bound to the surface domain. As such, the N211Q mutant would appear locked into a conformation that promotes generation of stable receptor dimers and simulates the activated state. Interestingly, the low affinity collagen-binding site in the DS domain faces a protruding loop in the DS-like domain that is very close to Asn211 (et al. (Recently, Carafoli et al.  describeet al. A. Collagm the KD , A and Bng event . A strucrylation . Thus, ie dimers  and helpo Asn211 . It is c (et al.  identifi (et al. . Althoug (et al. , resultiN-glycosylation.Previously, collagen I binding to DDR1 was shown to induce rapid receptor dimer aggregation and internalization in adherent cells, a series of events that are inconsistent with the reported slow kinetics of receptor activation . These fIt has been shown that both DDR1 and DDR2 display sustained activation after collagen stimulation, which can last for hours . This sl


Ten percent of the 250,000 proximal femur fractures that occur in the United States each year are malreduced into a varus position after treatment. Currently, there is no cephalomedullary nail available that allows the physician to dynamically change the lag-screw-to-nail angle. The Variable Angle Nail (VAN) was designed to allow movement of the lag screw relative to the shaft of the nail. This study compared the characteristics of the VAN to the Gamma 3 nail via finite element analysis (FEA) in stiffness and fatigue. The results of the FEA model with the same loading parameters showed the Gamma 3 and the VAN with lag-screw-to-nail angle of 120° to have essentially the same stiffness values ranging from 350 to 382 N/mm. The VAN with lag-screw-to-nail angles of 120°, 130°, and 140° should be able to withstand more than 1,000,000 cycles from 1,400 N to 1,500 N loading of the tip of the lag screw. The Gamma 3 should be able to last more than 1,000,000 cycles at 1,400 N. In summary, the VAN is superior or equivalent in stiffness and fatigue when compared to the Gamma 3 using FEA. More than 250,000 proximal femur fractures occur in the United States each year . Due to Unfortunately, despite incremental design advances over the last 15 years, surgeons still do not have a means of correcting intraoperative varus malreduction in situ when it occurs. Current implant designs incorporate a static lag-screw-to-nail angle curtailing the ability of the surgeon to change the reduction of the fracture once the implant is placed. The Variable Angle Nail  has been designed as a solution for the problem of intraoperative proximal femur varus neck-shaft angle malreduction. Because the lag-screw-to-nail angle can be changed intraoperatively to correct malreductions, one can choose the correct angle for the patient's anatomy. However, because the mechanism within the nail is unique, with multiple parts, and is dynamic rather than static, there is need to demonstrate the ability of the implant to withstand expected mechanical loads during clinical use of slightly more than 2 body weights –32. MoreThe purpose of this finite element analysis study was to describe the areas of high stress and location of failure in the VAN as compared to the Gamma 3 . Of special interest were areas of high stress concentration at the interface between the lag screw and the nail aperture and internal mechanisms for both the VAN and Gamma 3. Finite element analysis (FEA) of the VAN and Gamma 3 intramedullary nails was performed in several steps. Modeling the implants, testing parameters, material characteristics, static calculations, and fatigue calculations were performed as described in the following.The Variable Angle Nail (VAN) is comprised of a nail, a lag screw, and an internal mechanism that dynamically moves the lag screw from 120° to 140° of lag-screw-to-nail angles . The surd/D < 0.005 with contact and <0.0001 without contact.The FEA model for the VAN and Gamma 3 was built from a SolidWorks computer-aided design assembly . Autodesk Simulation software was used to build and analyze the model . The model consists of approximately 165,000 elements. A hybrid-meshing scheme was used in which 8-node brick elements comprise the majority of the volume. Fewer 6-node wedge, 5-node pyramid, and 4-node tetrahedral elements were used to close voids where needed. The material model accounted for plasticity in the nail, lag screw, and insert. The nail, lag screw, and insert were the only parts where yield occurred as a result of contact stresses. Surface-to-surface contact elements were employed between lag screw and nail, lag screw and insert, insert and nail, and insert and adjustment screw. The surface-to-surface contact was designed so that the nodes of either surface were prevented from passing through the nodes defining the other surface. A contact tolerance of 0.02 mm was used to define the gap value below which contact is assumed to occur. Frictionless contact was used, as vibration during cyclic testing was assumed to overcome frictional restraint between the parts. An adaptive contact stiffness algorithm was used by the FEA program to calculate contact stiffness. A nonlinear iterative solution method (Newton-Raphson) was used with line search Scheme. The Newton-Raphson method generates a search direction for new possible solutions, while the line search scheme is used to find a solution in the direction that minimizes the out-of-balance force error. The convergence criterion was displacement since the last time step. The convergence tolerance was ΔThree versions of this first model set were built with aperture angles of 120°, 130°, and 140° between the lag screw and the nail.A separate, second model of the interaction between the insert, adjustment screw, and nail was built and analyzed as well to delineate any high stress areas that may be transferred proximally from the lag screw to the insert and then to the adjustment screw and its threads that interact with the nail. This second one, or submodel, was created for the VAN design so that the stress levels in the threads and other features of the adjustment screw and its interface to the nail could be accurately modeled. Accurately modeling the contact elements in the threads of the adjustment screw to nail interface would have resulted in an unacceptably large model. In the full VAN model, contact elements are used between all the parts except the adjustment screw to nail interface and the nail to potting compound (both considered bonded). In the VAN model there were 12 sets of mating surfaces with contact elements.The adjustment screw was considered bonded to the nail in the first model of the VAN in order to focus on the interactions of the lag screw and the insert laterally and the lag screw and the nail medially. A third model (two versions) was created for the Gamma 3 nail with the aperture angles of 120° and 130° angles between the lag screw and the nail.Y-Z plane. The load was offset 50 mm from the lower axis of the nail . The stress-strain curve used in the FEA model was derived from a curve for annealed Ti-6Al-4V found in the Atlas of Stress-Strain Curves . The curSince the areas of high contact in the Gamma 3 and VAN nail are bearing type stresses, the local yield stresses may be exceeded without causing component failure.The Gamma 3 was tested at the worst-case scenario of 120° lag-screw-to-nail angle with a load of 1400 N directed distally on the lag screw. We chose the 120° because the resultant force vector loading the nail is more horizontal. Therefore, there is an increase in lever arm affecting the nail with an increased lag-screw-to-nail angle. The VAN was tested at 120°, with 1400 N load, and then at 120°, 130°, and 140° lag-screw-to-nail angles at 1500 N. We chose 1500 N because it is slightly more than two times average body weight. The stiffness plots for the VAN system were calculated using the 50 mm offset from the tip of the lag screw to the shaft of the nail. By necessity, the length of the lag screws is increased in length as the lag-screw-to-nail angle increased from 120° to 140°. The nail proximal bend angle of the Gamma 3 is 4° versus 6° for the VAN system resulting in a shorter lag screw moment arm length for the Gamma 3 (61 mm) versus the VAN (64 mm). The distance from the intersection of the lag screw axis from the center of the nail to the center of the load applicator was 64 mm, 72 mm, and 86 mm, respectively, for 120°, 130°, and 140° lag-screw-to-nail angles. For purposes of comparison, the stiffness values for both the VAN and Gamma 3 with 64 mm moment arms were used. The predicted stiffness for the Gamma 3 with a 64 mm moment arm is 350 N/mm between 0 and 1500 N.The measured test results for the stiffness of the VAN system from 0 to 1500 N were 340 N/mm for a 120° lag screw angle and an average value of approximately 310 N/mm for 130° and 140° lag screw angles. The level of static tensile stress that should not be exceeded if 1,000,000 load cycles were to be achieved before failure was chosen to evaluate the fatigue capability of the VAN and Gamma 3 designs see .The data for Ti-6AL-4V lists a fatigue strength of 74,000 psi (510 MPa) at 10,000,000 cycles, and the ultimate strength is 131,000 psi (900 MPa) , 36. BecThe potential for compressive fatigue was further evaluated by comparing the von Mises stresses with the maximum and minimum principal stresses in the regions of high compressive loading in the nail and insert. The von Mises stress is most commonly used in noncyclic FEA as the best prediction of the material yield point in a multiaxial stress state. The von Mises calculation utilizes distortion energy to find an equivalent value of uniaxial stress, which can be compared to yield point test data. In fatigue analysis, the failure limits in tension and compression can be quite different. Loading in compression can be significantly higher than loading in tension to achieve the same fatigue lifetime.  The stress state in the VAN and Gamma 3 analysis are comprised of an average stress and a range stress. In the Soderberg diagram , the aveTherefore, for a fatigue life of 1,000,000 cycles, Se = 566 MPa, and solving for Smax, the maximum tensile stress of the FEA plots should be less than 686 MPa. Moreover, for a fatigue life of 200,000 cycles, the maximum stress on the FEA plots should be less than 711 MPa. In other words, a peak tensile stress of 686 Mpa should not be exceeded at the maximum principal stress areas located on the medial and lateral parts of the nail and nail-insert. This location, the aperture for the lag screw in the Gamma nail, has been found to be the most common location of nail breakage , 39. TheThe FEA model illustrated that the 120° Gamma 3 and the 120° VAN had essentially the same stiffness values of 350 N/mm and 340 N/mm, respectively . However Based on the fatigue formula calculations described previously, the peak tensile stress derived by the FEA should not exceed 680 Mpa. At 1400 N of loading, with the VAN set to 120° lag-screw-to-nail angles, the maximum stress was 500 MPa at the proximal aspect and 620 MPa at the distal aspect of the lateral aperture . A GammaThis study was performed to evaluate the mechanical properties of the VAN relative to the Gamma 3. Finite element analysis was performed in order to evaluate the stresses that are transferred from the lag screw onto the VAN during normal loading conditions as compared to the Gamma 3 nail. The analysis suggests that the VAN internal mechanism and nail have a decrease in stresses when compared to the Gamma 3 during nearly similar loading conditions (1400 N and 1500 N). Typically, Ti alloy nail implants placed in the proximal femur to fix hip fractures fail in tension at the distal aspect of the lateral lag screw aperture . The allGiven the expected increasing number of proximal femur fractures, variety of anatomic variations in neck-shaft angles and the associated prevalence of varus malreduction following cephalomedullary nailing , 10, 11,The limitations of this study may be due to implant modeling, testing set-up assumptions, or computational testing analysis. When designing this study, we sought to minimize any implant modeling error by developing virtual nails from actual implants. The high mesh density of the FEA models resulted in close approximations of the actual nail systems studied. The models consist of approximately 165,000 solid elements, the majority of which are 8-node bricks. In a few regions where 8-node bricks would result in an unacceptably high mesh density, 6-node wedge, 5-node pyramid, and 4-node tetrahedral elements are used to close voids in the model. Material properties were accounted for by allowing for plasticity in the nail, lag screw, and insert. In clinical practice, the lag screw tends to slide within the nail aperture in patients that are treated with these types of implants. As the lag screw slides, the lever arm decreases and minimizes the stresses seen within the nail. The worst-case situation was emulated by holding the lag screw to the nail by spring elements that did not allow the lag screw to slide. This could change the mechanical environment slightly. However, since the nails were matched to each other, this effect was probably minimized in the comparison between them. By following protocols commensurate with previous studies that evaluated the stiffness and fatigue of implants of this nature, the testing set-up error was minimized. Moreover, established mechanical and FEA testing parameters that exist for proximal femoral fracture nails were employed in order to create uniform testing .One limitation of this study is that there is no previously published mechanical testing of the VAN system. Moreover, there is no in vivo analysis of strain, or even FEA, of the Gamma 3 nail in the unstable hip fracture model that we are aware of. Therefore, although the authors tried to test these two implants relative to each other, the applicability to clinical use is not established as of yet.In summary, the finite element analysis comparing the Variable Angle Nail (VAN) to the Gamma 3 nail illustrated that there are minimal differences in stiffness or fatigue between the two nails but with the potential advantage changing dynamically the angle between the lag screw and accommodating multiple patient multiple femoral neck-shaft angles with a single nail. Future mechanical testing of strength and fatigue should be performed to further evaluate the feasibility of using the VAN in clinical practice.


N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the  This modification helps the EC to express the middle and late genes of lambdoid phages by suppressing many Rho-dependent and -independent terminators present on the phage DNA ,11. N isnut site , and sub factors . The C-t factors  near the factors , which m factors . This co factors .Figure nutR site by forming a N-NusA-Rho ternary complex, which renders slow rate of ATP hydrolysis of the former; (ii) exerts anti-termination most likely by modifying the RNA exit channel of the EC, which is operational even far away from the nut site; and finally (iii) removes NusA from the Rho-dependent termination path.The mechanism of N-mediated suppression of RNA hairpin-dependent termination has been studied in detail ,18,19. HSupplementary Table S4. All the in vivo anti-termination assays were performed in different derivatives of Escherichia coli rac− strain MC4100. The strains GJ5147 and RS445 used in β-galactosidase assays contain single-copy Plac-H-19B nutR/tR1- lacZYA (GJ5147) or Plac-lacZYA (RS445) reporter cassettes as λRS45 lysogen. Strain RS1017 was constructed by moving Plac– H-19B nutR/tR1- trpt'-lac ZYA reporter cassette by λRS45 mediated transduction from pRS992 into the strain RS257. This construct has two terminators tR1 and trpt' attached sequentially. Strain RS1018 and RS1019 were also constructed by moving Plac-H-19B nutR/tR1-tR’-lacZYA and Plac-λnutR/λtR1–lacZYA, respectively, in the same way into RS257. Temperature-sensitive (ts) allele of rpoC [rpoCR120 (ts)] was moved to RS445, RS734 and RS1017, resulting in strains RS940, RS941and RS1029, respectively, by P1 transduction. Plasmid pRS1092 was constructed by inserting trpt' sequence at the SmaI site present after the nutR/tR1 sequence of pRS22 (pTL61T with pT7A1– H-19B nutR-tR’-T1T2-lacZYA) to make the double terminator, tR1-trpt', construct. This trpt' sequence was amplified from pRS992 using oligos RS567/RS568. XL-red strain (Stratagene) was used for random mutagenesis . The strains RS734 and RS445 were transformed with the plasmids having the mutants and wild-type (WT) H-19B N genes to estimate the in vivo anti-termination efficiency at the H-19B tR1 rho-dependent terminator (tR1-trpt’ terminators (tR’ (Supplementary Table S1), respectively. The strains RS1019 and RS445 were transformed with the plasmids having mutant and WT λ N genes to estimate the in vivo anti-termination efficiency at λtR1 rho-dependent terminator . In this case, strains were grown at 42°C, to inactivate the chromosomal copy of the WT rpoC gene  were transformed with the suppressor and the WT Rho plasmids, and were subsequently made Δrho in the chromosome by P1 transduction (Supplementary Table S2).To estimate the sduction A. RatiosAll the measurements of β-galactosidase activities were done in a microtiter plate using a Spectramax plus plate reader following the published procedure ,22.In vitro Rho-dependent termination reactions were performed in T buffer (25 mM Tris–HCl (pH 8.0), 5 mM MgCl2, 50 mM KCl, 1 mM DTT and 0.1 mg/ml of BSA) at 32°C. The reactions were initiated with 10 nM DNA, 40 nM WT RNAP, 175 μM ApU, 5 μM each of GTP and ATP and 2.5 μM CTP to make a 23-mer EC23. [α- 32P]CTP (3000 Ci/mmol) was added to the reaction to label the EC23. The complex was chased with 250 μM NTPs in the presence of 10 μg/ml of rifampicin for 15 min at 32°C. Also, 50 nM WT Rho, 200 nM NusG, 300 nM NusA and 100 nM WT or mutant H-19B N were added to the chase solution as indicated. The reaction products were separated on 8% sequencing gels and analysed by phosphorimager . Preparations of N, NusA and Rho proteins and the DNA templates are described in the supplementary methods.orimager , 6 and 7emplates  were don2PO4 (pH 3.5) as the mobile phase. In all the assays, the composition of the reaction mixture was 25 mM Tris–HCl (pH 8.0), 50 mM KCl and 5 mM MgCl2, 1 mM DTT and 0.1 mg/ml of BSA. Assays were performed on the nascent RNA emerging out of the transcription EC. Stalled ECs were formed at the lac operator site on the T7A1-nutR/tR1-lacO or T7A1-nutR/tR1-trpt'-lacO templates in the same way as described earlier. These complexes were incubated with 100 nM Rho in presence of 1 mM NTPs and [γ-32P]ATP (3000 Ci/mmol). Aliquots were removed and mixed with 1.5 M formic acid at various time points to stop the reaction. Release of Pi was analysed by exposing the TLC sheets to a Phosphorimager screen for 5 min and subsequently by scanning using Typhoon 9200 (Amersham), and the intensities of ATP and Pi were quantified by Image QuantTL software .ATPase activity of the WT Rho protein was measured from the release of inorganic phosphate (Pi) from ATP after separating on the polyethyleneimine (PEI)-cellulose TLC plates (Merck) with 0.75 M KH0 + a/[1 + exp {-(x − x0)/λ}]. In these equations, ‘λ’ denotes the rate and ‘a’ is the amplitude. The r2 values for each of the fittings were ≥0.99. For rate calculations, only the initial slopes were considered as later points in the curves originated from multiple rounds of ATP hydrolysis by Rho after dislodging the EC. The initial slopes of the plots give ATPase activity of Rho in terms of nanomoles of ATP hydrolysed per minute (Amounts of ATP hydrolysed (in nanomoles) were plotted against time using SIGMAPLOT software. The data points for all the curves, except for +N, were fitted to the equation of an exponential curve, y = a*[1-exp(-λ*x)]. The data points obtained for ‘+N’ experiments values were fitted to a sigmoidal equation; y = yr minute ,21.For the ATPase assays of E134K using poly(C), 50 nM Rho was incubated with 1 mM ATP, together with [γ-32P]ATP  at 37°C, and ATP hydrolysis was initiated by the addition of 20 µM poly(C). Products were analysed by the same way as described earlier. The initial rates of the reaction were determined by plotting the amount of hydrolysed ATP versus time using linear regression method D.nutR boxA, spacer, boxB and the region immediately after boxB, respectively, were used. Stalled EC was formed at the lacO site of the template pT7A1-nutR-lacO-tR’ immobilized to the magnetic beads, in the same way as described earlier, in the presence of N, NusA and NusG. The EC was washed to remove free NTPs and was then incubated with 50 nM Rho in presence of 1 mM AMPPNP. In all, 10 μM of each of the anti-sense oligos were then added to the reactions for 30 sec, following which one unit RNase H was added and incubated for 1 min at 32°C. The reaction was stopped by extracting with phenol, mixed with equal volume of form amide loading dye and loaded onto an 8% sequencing gel , and incubation was continued for 30 min at 25°C. Excess SPDP was removed by passing the mixture through a protein-desalting column (Pierce). In all, 100 nM of SPDP-derivatized Rho was then added to the stalled EC, which was formed in a similar way as in footprinting experiments using the same DNA template, except that RNA was not radio-labelled, instead P32-labelled WT NusA was used. WT NusA has three cys residues (one is in KH1 domain and other two are in AR2 domain). After formation of the stalled EC in the presence of NusA and N, it was washed by phosphate buffer, followed by incubation for 10 min at 37°C with 100 nM of SPDP-labelled WT or Y80C Rho. SPDP-Rho-N cross-linking was not attempted because we observed very high level of non-specific adsorption of N onto the streptavidin beads (Promega), which might had led to spurious cross-linked products outside the EC. Reactions were stopped by non-reducing SDS-sample dye (-βME) and loaded onto a non-reducing 6–10% gradient PAGE, and the products were analysed in a Fuji phosphorimager -propionamido] hexanoate (Pierce), which cross-links cysteine and amine. We first labelled the surface-exposed primary amines (from lys residues) of a no-cysteine derivative of WT (C202A) and Y80CRho (C202A). The mutant Y80C Rho could not be made ‘zero’ cys to maintain the Y80C mutation. This derivative of Rho has 28 lys residues against one cys (at the position 80) per monomer; therefore, on SPDP labelling, majority of the modifications will be in the lysines, and it is likely that cysteins will not be labelled. The concentrations of the cross-linker and Rho were 5 μM and ∼18 μM , respectively. Concentration of the cross-linker was kept less to get a sub-saturating labelling of Rho, which ensured the absence of non-specific cross-linking in the subsequent steps. The lowest concentration of SPDP was determined by trials. Rho and SPDP were mixed in phosphate buffer (100 mM NaHorimager E and F.nutR site (on the right operon) of the lambdoid phages and the rut site of the tR1 Rho-dependent terminator on the nascent RNA overlap with each other  does not have an overlapping Rho-dependent terminator, and hence we focussed only on the nutR site.The ch other ,24. When earlier ,26. The nutR site, N-NusA-Rho forms a ternary complex by co-occupying the same site. In this ternary complex, Rho may get fully inactivated or its conversion into a translocase-competent form becomes slow  present as a λRS45 lysogen in a lac− strain of MC4100 . This nutR/tR1 sequence has the overlapping N (the nut site) and the Rho binding sites (the rut site), and it is derived from a lambdoid phage H-19B . R15C, R15P and R18P mutations are part of the ARM (..12RSRRRER18..), which recognizes the nut site. Anti-termination assays  revealed that these mutations were severely defective on this single terminator construct  may not be functionally important for anti-termination on this construct. Dispensability of the c-terminal 14 amino acids of the λN protein was observed earlier . But, the ΔCTD N (Δ101–127) was able to show significant level of anti-termination  downstream of the nutR site. Similar to the single terminator construct, this one was also inserted into the chromosome as a lysogen . In this construct, Rho-entry site in the second terminator will not face any interference from the N binding, as it is devoid of nut site. We repeated the in vivo anti-termination assays with WT and different N mutants on this construct. On this template, point mutants in the ARM region remained severely defective for the anti-termination as before, but the CTD deletion mutants, which were largely unaffected on the single terminator construct, were now significantly defective (Supplementary Table S1). Similarly, the RNAP mutants also showed defect . Lac-operator sequence was fused either next to the nutR/tR1 terminator to stall the EC near the nut site , and this was comparable with those observed earlier from other RBs (nut site. These results showed that (i) the N-nut interaction is more important for preventing Rho to act on the stalled ECs near the nut site, whereas N-RNAP interaction is equally important for preventing the RNA release from the ECs stalled further away from the nut site; and (ii) if sufficient time is allowed, Rho is capable of overcoming the inhibition/anti-termination function of N. This stabilization of EC away from the nut site is similar to that observed with the stalled EC at a terminator hairpin  N slows down the initiation of ATP hydrolysis and the translocase activity of Rho. We tested the effect of N bound to the nut site on the ATPase activity of Rho. We have used the nascent RNA attached to the stalled ECs . These results indicated that the effect of N on the ATPase activity of Rho is confined near or at the nut site.The observation that N reduces the rate of RNA release by Rho E and F aSupplementary Figure S3A and B; nut/rut site involves slowing down of the initiation of the ATP hydrolysis by Rho.Further, we hypothesized that this inhibitory effect of N can be overcome by increasing the rate of ATP hydrolysis of Rho. We used a Rho mutant, P235H, with a higher rate of ATP hydrolysis . Rho + AMPPNP  was added to this stalled EC. We have earlier observed that AMPPNP-bound Rho interacts specifically with the spacer region of the nut/rut site with a small footprint (∼22 nt), which most likely reflects the initial RNA-loading step of Rho .We stalled the EC in the presence and absence of N by lac repressor bound at a p of Rho . This fospacer and boxB regions of the nutR/rut site . RNase T1 cleaves at the single-stranded G residues. Owing to the presence of two hairpins in the nutR site  and boxB . Addition of Rho to this N-modified EC did not change the protection pattern. Consistent with our earlier observations  were protected when N +NusA/NusG were present , which specifically forms inter-molecular cross-links between primary amines (e.g. lysines) of a cysteine-less protein, C202A Rho  and the cysteine side-chains of NusA (having three cysteines). The amine-cysteine Rho-NusA cross-linked product can be identified on a non-reducing SDS-PAGE using radiolabelled NusA and by comparing with appropriate molecular weight markers.To establish the co-occupancy of N, NusA and Rho at the ex as in A. We havnut/rut site of the EC, which is stalled 90 nt downstream of the boxB hairpin. This result strongly supports the proposal of co-occupancy of Rho-N-NusA and the possibility of Rho-NusA interaction at the nut/rut site.For the cross-linking experiments, similar RB complexes as described in nut/rut site by using a direct binding assay of the former to the RB described in Supplementary Figure S7A; Supplementary Figure S7B). This association of Rho was through the nut/rut site of the nascent RNA because a Rho mutant, Y80C . We used the template described in lac operator site and the anti-termination activity of N at the hairpin-dependent terminator tR'. We, at first, made stalled ECs in the presence of lac repressor , and they were capable of elongation when IPTG  was added in the absence of Rho . The N-modified stalled EC was also observed to read through the tR' terminator efficiently to yield the RO product (lane 9). In the absence of N, Rho terminated the stalled EC, which was evident from the accumulation of the RB product over time . In the presence of N, not only anti-terminated products (RO) were formed but also a slow accumulation of RB at the lac operator site was observed . These results suggested that Rho can still terminate in the presence of N albeit with a slower rate, which further confirmed the co-occupancy of N, NusA/G and Rho factors at the nutR/rut site.Finally, we probed the functional consequences of the co-occupancy of N and Rho on the rho gene and screened for mutants, which retained the termination function even in the presence of WT N (see Supplementary Methods). We isolated a Rho mutant, E134K. Another Rho mutant, P103L, was reported to prevent growth of a certain λ phage, λr32, probably by overcoming the N function .To identify whether any other steps in Rho-dependent termination is affected by N, we looked for suppressor mutations in Rho, which enable it to overcome N. We randomly mutagenized the function ,35,36. Win vitro anti-termination activities of H-19 B N in the presence of the E134K Rho using the H-19B nutR/tR1 terminator template . Next, we investigated the in vitro termination efficiency of E134K Rho both in the presence and absence of NusA and NusG. We used two separate templates, nutR/tR1 or trpt' terminators fused to the T7A1 promoter . In the absence of any factor, E134K Rho showed termination defect on both the terminators . Unlike WT Rho, NusG on its own was unable to induce early termination or improve the efficiency of E134K Rho (lane 8 of 7A and lane 13 of 7B). Interestingly, instead of delaying the termination, NusA improved the termination efficiency and together with NusG, made E134K Rho extremely efficient and early terminating .At first, we measured the promoter A and B. Supplementary Figure S9A and B). NusG–Rho interaction stimulates the termination process by increasing the speed of RNA release and does not affect either RNA-binding or RNA-dependent ATPase activities of Rho .Therefore, we, at first, tested the RNA-binding and RNA-activated ATPase activity of E134K Rho. We assessed the RNA binding of E134K by gel-shift assays, using a short RNA, rCrut site . Comparerut site C. It alsrut site D. These The defects described earlier may give rise to a slow rate of RNA release by E134K from a stalled EC, and NusA may improve this rate. We measured the RNA release by E134K from a stalled EC, similar to the one described in nut/rut site . They interact with the nut site on mRNA . Hence, we concluded that N functions independent of Rho-NusG CTD interaction. However, incorporation of NusG into the N-anti-termination machinery can alter NusG-NTD-β' clamp helices interactions, which in turn may perturb the Rho-NusG complex formation.NusG stimulates N activity in vitro  and was  process . On the  process ,40, and  binding  did not The small anti-terminator protein N has three interacting regions ; Government of India and intramural funding of Centre for DNA Fingerprinting and Diagnostics (CDFD); Swarnajayanti fellow of Department of Science and Technology, Government of India (to R.S.); senior research fellowship for DBT (to G.M.); post-doctoral fellowship (to D.D.); Indian Council of Medical Research (ICMR) senior research fellowship (to S.M.). Funding for open access charge: Waived by Oxford University Press.Conflict of interest statement. None declared.


H2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract.Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (T Cryptomeria japonica) pollinosis is one of the most serious allergic diseases in Japan,  at room temperature for 2 hours. The sera were then applied onto a previously prepared n-CPA9-coated microtiter plate for testing IgE cross-reactivity. Subsequent secondary antibody staining and signal detection was carried out as described above.cpa9 cDNA composed of 2562 nucleotides encoding 757 amino acids, which was homologous to subtilisin-like serine protease family (data not shown). Its calculated molecular weight and isoelectric point were 80148.79 Da and 6.22, respectively. The CPA9 sequence contained the catalytic triad  responsible for the proteolytic activity of subtilisin-like serine proteases [Our comprehensive 2-D IgE immunoblotting study provided a compelling evidence for the presence of 31 unidentified major allergen spots that showed a higher IgE-binding frequency than that of Cry j 2 (40%) . Among troteases . Intriguroteases  which waE. coli cold shock expression system. SDS-PAGE and anti-His immunoblotting analyses revealed a successful soluble expression of 145 kDa r-CPA9-TF fusion protein. We purified this fusion protein using His-Trap chelate affinity chromatography, and nonfusion r-CPA9 (seen as a single band protein under silver staining of a SDS-PAGE gel) was prepared by thrombin digestion and further purification by nonreducing SDS-PAGE and gel extraction procedures (data not shown). We also prepared murine CPA9-specific antisera upon immunization with the purified r-CPA9.We next tried to produce r-CPA9 using C. japonica pollen extract. Two successive chromatographic steps on a HiTrap Butyl HP hydrophobic interaction column and a Mono Q anion exchange column yielded a single 90 kDa protein as seen under reducing SDS-PAGE analysis (data not shown). Immunoblotting also indicated that the obtained 90 kDa protein was recognized by anti-CPA9 polyclonal antibody. Furthermore, TOF-MS/MS analysis demonstrated that 2 internal amino acid sequences of the 90 kDa antigen were completely coincided with those deduced from cpa9 cDNA. Taken together, we concluded that the purified 90 kDa molecule was n-CPA9.For further immunochemical characterization of CPA9, we next sought to obtain n-CPA9 from To examine IgE-binding frequency of n-CPA9, we next performed ELISA using sera from 26 Japanese cedar pollinosis patients (RAST score ≥2) and 5 healthy donors as a control. We found that n-CPA9 showed 88.5% IgE binding frequency (23/26), which was higher than that observed against major allergen Cry j 1 . This suggests that CPA9 is an additional major Japanese cedar pollen allergen.μg/ml melon extract, and preincubation with 10 μg/ml extract completely inhibited IgE-binding. We also found that C. japonica pollen extract  showed a nice inhibitory action, whereas no competition was observed upon incubation with OVA (negative control). These results raise a possibility that this subtilisin-like serine protease might be a new class of plant cross-reactive allergen, although actual IgE-cross-reactivity to Cuc m 1 and to other subtilisin-like allergens needs to be confirmed in future studies.Because CPA9 polypeptide was highly homologous to the melon subtilisin-like endopeptidase allergen Cuc m 1,  we next In summary, we identified a novel class of Japanese cedar pollen allergen (CPA9) homologous to the plant subtilisin-like serine proteases. Its higher IgE-binding frequency than that of the major allergen Cry j 1 suggests that this molecule should be an additional important allergen in Japanese cedar pollinosis, although further analyses are needed to better characterize its allergenicity and proallergic potency. We believe that CPA9 would be applicable for the development of component-resolved diagnosis and specific immunotherapy of Japanese cedar pollinosis. Furthermore, this molecule offers a good model to assess a possible role of serine protease activity in the pathogenesis of cedar pollinosis.Ahmed Ragaa Nour Ibrahim and Seiji Kawamoto contributed equally to the work.