Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs).Although thrombin is a recognized physiological activator of PAR 1 and PAR 4 , the endogenous enzymes responsible for activating PAR 2 in settings other than the gastrointestinal system, where trypsin can activate PAR 2 , are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR 1 , PAR 2 , and PAR 4 ; 2) PAR-dependent calcium signaling responses in cells expressing PAR 1 , PAR 2 , and PAR 4 and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR 4 -dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR 1 , PAR 2 , and PAR 4 and to disarm/inhibit PAR 1 . Although hK5 and -6 were also able to activate PAR 2 , they failed to cause PAR 4 -dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR 2 -mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR 1 , PAR 2 , and PAR 4 . Proteinase-activated receptors (PAR 1-4 )3 compose a unique family of four G-protein-coupled cell surface receptors for certain proteinases (1-9). Proteolytic cleavage within the extracellular N terminus reveals a tethered ligand that binds to the extracellular receptor domains to initiate cell signaling (5, 6, 9, 10). Proteinases that activate PARs include coagulation factors, enzymes from inflammatory cells, and proteinases from epithelial cells and neurons. These enzymes, generated and released during injury and inflammation, can cleave and activate PARs on many cell types from a variety of species (humans, rats, and mice) to regulate the critically important processes of hemostasis, inflammation, pain, and tissue repair. Other proteinases that cleave PARs downstream of the N-terminal tethered ligand sequence disable the receptors for further proteolytic activation, thus abrogating PAR signaling. It is of considerable interest to identify the proteinases that activate and/or disable PARs, in view of the emerging role that these receptors can play in diseases such as asthma, arthritis, inflammatory bowel disease, and cancer (5-7).In some systems, the proteinases that activate PARs have been established. For example, in the circulatory system, PAR 1 , PAR 3 , and PAR 4 are recognized physiological targets for thrombin (4), which does not efficiently acti...

We have identified a novel serine protease, myelencephalon-specific protease (MSP), which is preferentially expressed in the adult CNS, and therein, is abundant in both neurones and oligodendroglia. To determine the potential activity of MSP in CNS demyelination, we examined its expression in multiple sclerosis lesions and in two animal models of multiple sclerosis: Theiler's murine encephalomyelitis virus (TMEV) and myelin oligodendrocyte glycoprotein (MOG)-induced experimental allergic encephalomyelitis (EAE) in marmosets. High levels of MSP were present within infiltrating mononuclear cells, including macrophages and T cells, which characteristically fill sites of demyelination, both in multiple sclerosis lesions and in animal models of this disease. The functional consequence of excess MSP on oligodendroglia was determined in vitro by evaluating the effects of recombinant MSP (r-MSP) on oligodendrocyte survival and process number. Application of excess r-MSP resulted in a dramatic loss of processes from differentiated oligodendrocytes, and a parallel decrease in process outgrowth from immature cells. Transfection of oligodendrocyte progenitors with an MSP-green fluorescent protein construct produced similar changes in oligodendrocyte process number. Importantly, r-MSP did not affect oligodendrocyte survival or differentiation towards the sulphatide-positive lineage. We further demonstrate that myelin basic protein, and to a lesser extent myelin oligodendrocyte glycoprotein, can serve as MSP substrates. These studies support the hypothesis that excess MSP, as is present in inflammatory CNS lesions, promotes demyelination.

The human kallikreins are a large multigene family of closely related serine-type proteases. In this regard, they are similar to the multigene kallikrein families characterized in mice and rats. There is a much more extensive body of knowledge regarding the function of mouse and rat kallikreins in comparison with the human kallikreins. Human kallikrein 6 has been proposed as the homologue to rat myelencephalon-specific protease, an arginine-specific degradative-type protease abundantly expressed in the central nervous system and implicated in demyelinating disease. We present the xray crystal structure of mature, active recombinant human kallikrein 6 at 1.75-Å resolution. This high resolution model provides the first three-dimensional view of one of the human kallikreins and one of only a few structures of serine proteases predominantly expressed in the central nervous system. Enzymatic data are presented that support the identification of human kallikrein 6 as the functional homologue of rat myelencephalon-specific protease and are corroborated by a molecular phylogenetic analysis. Furthermore, the xray data provide support for the characterization of human kallikrein 6 as a degradative protease with structural features more similar to trypsin than the regulatory kallikreins.Recent studies demonstrate that humans have a large multigene family of at least 15 different kallikreins (serine type proteases, abbreviated as KLK 1 in reference to the gene, or hK in reference to the protein) (1). Similarly, the mouse and rat kallikrein gene families are characterized by a large number of closely related members that presumably arose because of gene duplication events (2-6). The different members of the mouse and rat kallikreins are characterized by a high degree of amino acid identity, but typically exhibit different preferences toward peptide substrates (7-12). Several human kallikreins have been identified as potentially useful diagnostic markers for breast (KLK3 and KLK6), prostate (KLK2 and KLK3), and ovarian (KLK6, KLK9, KLK10, and KLK11) cancers as well as neurodegenerative diseases such as Alzheimer's (KLK6) (1, 13-17).Myelencephalon-specific protease (MSP) is a member of the rat kallikrein gene family that is abundantly expressed in the rodent central nervous system and shown to be up-regulated in response to glutamate receptor-mediated excitotoxic injury (18). Potential human homologues to rat MSP have also been identified (18) and have been alternatively named protease M (19), Zyme (20), and neurosin (21). Mouse homologues to MSP have been reported as brain and skin serine protease (BSSP) (22) and brain serine protease (BSP) (23). It has been postulated that MSP/protease M/neurosin may play a key role in the regulation of myelin turnover and in demyelinating disease (18, 24 -27), including the development of multiple sclerosis lesions (25). Furthermore, this kallikrein may also play a role in the degradation of ␤-amyloid or turnover of amyloid precursor protein (28,29). The kinetic properties of MSP have ident...

A full-length cDNA clone of a previously unidentified serine protease, myelencephalon-specific protease (MSP), has been isolated by using a PCR cloning strategy and has been shown to be expressed in a nervous system and spinal cord-specific pattern. Sequence analysis demonstrated that MSP is most similar in sequence to neuropsin, trypsin, and tissue kallikrein and is predicted to have trypsin-like substrate specificity. MSP mRNA was found to be ϳ10-fold greater in the CNS of the rat and human, as compared with most peripheral tissues, and within the CNS was found to be highest by a factor of four in the medulla oblongata and spinal cord. Levels of mRNA encoding tissue plasminogen activator (tPA) also were elevated in the spinal cord but were more widespread in peripheral tissues as compared with MSP.In the adult rat lumbosacral spinal cord, in situ localization of MSP mRNA demonstrated 2-fold higher levels in the white, as compared with the gray, matter. MSP mRNA expression was shown to increase 3-fold in the white matter and 1.5-fold in the gray laminae at 72 hr after intraperitoneal injection of the AMPA/ kainate glutamate receptor-specific agonist, kainic acid (KA). MSP mRNA remained elevated in the ventral gray matter, including expression associated with the motor neurons of lamina IX, at 7 d after the initial excitotoxic insult. Together, these observations indicate that MSP is in a position to play a fundamental role in normal homeostasis and in the response of the spinal cord to injury.