The human proenkephalin A gene (PENK-A) contains 4 exons and codes for a series of structurally related oligopeptides like methionine enkephalin (Met-ENK), leucine enkephalin (Leu-ENK), methionine enkephalin arginine phenylalanine (Met-ENK-Arg-Phe) as well as methionine enkephalin arginine glycin leucine (Met-ENK-Arg-Gly-Leu), wherein one molecule of proenkephalin comprises the sequences of 4 Met-ENK and one Leu-ENK, Met-ENK-Arg-Phe and Met-ENK-Arg-Gly-Leu each. Met-ENK-Arg-Phe and Met-ENK-Arg-Gly-Leu are further metabolised to Met-ENK. The enkephalins have a function as neurotransmitters as well as neuromodulators and neurohormones. PENK-A besides the enkephalins comprises the sequences of enkelytin and peptide B at its N-terminus as well as synenkephalin at its C-terminus, that have an antibacterial function.
The expression of proenkephalin occurs in the central nervous system as well as the peripheral nervous system. In the brain increased concentrations of PENK are observed in the nucleus caudatus and the nucleus accubens, in the peri-aqueductal grey as well as in the hippocampus and the raphe nuclei.
Enkephalins play a major role in a variety of physiological processes like perception of pain, regulation of stress response comprising hormone regulation, regulation of bone formation as well as regulation of immune responses. Met-ENK stimulates the proliferation of B- and T-lymphocytes and Leu-ENK the proliferation of T-helper and cytotoxic T-cells. Due to the immune regulatory properties of Met-ENK this enkephalin is classified as cytokine. Endogenous enkephalin peptides are also involved in normal regulation of cardiovascular functions like heart frequency, contractile strength and arterial blood pressure.
Enkelytin has antibacterial function against gram-positive bacteria like Streptococcus aureus, Bacillus megaterium and Micrococcus luteus, however it is not inhibiting the growth of gram-negative bacteria like Escherichia coli, as well as the growth of fungi. Synenkephalin shows antibacterial properties against gram-positive bacteria as well as gram-negative bacteria.
The level of enkephalin is altered in body fluids and tissue in a variety of diseases. The Met-ENK immune reactivity in plasma of patients suffering from a brain infarctus in the acute phase and diabetes patients with symptomatic myocardiac ischemia is significantly increased as compared to healthy control individuals, as well as in plasma and liquor of patients suffering from migraine and headache and in liquor of schizophrenia patients.
In Parkinson's disease the content of Met-ENK-Arg-Gly-Leu in liquor of patients is significantly decreased as compared to healthy control individuals.
Patients suffering from Alzheimer's disease showed a 4-fold increase in Met-ENK-like immune reactivity.
The concentration of enkephalin m-RNA in the striatum of patients suffering from Huntington's disease is significantly reduced, in liquor a decrease of met-ENK-Arg-Gly-Leu concentration was shown.
In tumor diseases prolactin-(PL) and adrenocorticotropin (ACTH)-secreting adenomas of the hypophysis show an increased concentration of Met-ENK as well as a 10-fold increased concentration of proenkephalin peptides in the latter.
Enkephalins play a role in the pathophysiological response of nerve tissue in traumatic injuries like head injuries and injuries of the skin for example during and after surgery.
Enkephalins furthermore play a major role in the pathogenesis of systemic inflammations. In animal models the induction of a peritonitis or the application of lipoypolysaccarides lead to an increase in PENK-expression as well as to an increase in Met-ENK-plasma concentrations.
In abscesses of periarthritic cattle different PENK-fragments could be detected, that comprise the sequence of enkelytin. In human peripheric monocytes and rat lymphocytes the induction of PENK-m-RNA expression by lipopolysaccharides could be demonstrated:
The infection of rats by borna-viruses resulted in an increased PENK-A transcription in the striatum.
Met-ENK, Met-ENK-Arg-Phe and the proenkaphaline peptides B and F are significantly increased in plasma during hemorrhagic hypotension.
The biosynthesis of enkephalins (see FIG. 2) occurs as is the case in other peptide hormones, as preprohormone at (Golgi-) bound ribosomes. After separation of the hydrophobic N-terminal signal sequence by so-called signal peptidases and folding of the proteins in the lumen of the endoplasmic reticulum, the propeptides are packed into vesicles in the Golgi apparatus and are transported to the cell membrane. During transport the propeptides are processed to mature hormones by so-called prohormone convertases at usually dibasic amino acid sequences. Via different stimuli the peptides are secreted into the extracellular space or into the plasma. The mature peptides are rapidly inactivated after secretion by proteolysis.
Enkephalins have a half-life of 12 to 15 minutes in plasma (ex vivo).
The processing of proenkephalin occurs in several steps in a strict timely order and is tissue-specific. An earlier step in processing is the separation of the C-terminal peptide B that comprises the sequences of enkelytin and Met-ENK-Arg-Phe. The processing of proenkephalin leads to the formation of different proenkephalin peptides.
The presence of the PENK-fragment 119-159 in liquor, however not in blood, was already shown by Stark et al., 2000.
As this fragment plays a central role in this invention, it is called “enkephalin-footprint” (ENK-fp) herein.