Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only ∼0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved C3i 10- to 20-fold faster, but still at only ∼1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes. Nevertheless, relative rates of reaction with α-2-macroglobulin and C1-inhibitor suggest that α-2-macroglobulin may be a significant physiological inhibitor of MASP-1.

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Demyelination, the proteolytic degradation of the major membrane protein in central nervous system, myelin, is involved in many neurodegenerative diseases. In the present in vitro study the proteolytic actions of calpain, human trypsin 1 and human trypsin 4 were compared on lipid bound and free human myelin basic proteins as substrates. The fragments formed were identified by using N-terminal amino acid sequencing and mass spectrometry. The analysis of the degradation products showed that of these three proteases human trypsin 4 cleaved myelin basic protein most specifically. It selectively cleaves the Arg79-Thr80 and Arg97-Thr98 peptide bonds in the lipid bound form of human myelin basic protein. Based on this information we synthesized peptide IVTPRTPPPSQ that corresponds to sequence region 93-103 of myelin basic protein and contains one of its two trypsin 4 cleavage sites, Arg97-Thr98. Studies on the hydrolysis of this synthetic peptide by trypsin 4 have confirmed that the Arg97-Thr98 peptide bond is highly susceptible to trypsin 4. What may lend biological interest to this finding is that the major autoantibodies found in patients with multiple sclerosis recognize sequence 85-96 of the protein.Our results suggest that human trypsin 4 may be one of the candidate proteases involved in the pathomechanism of multiple sclerosis.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.