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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Mannose- or mannan-binding lectin (MBL) is a member of the collectin protein family, which includes lung surfactant proteins SP-A and SP-D. Each member consists of similar or identical polypeptide chains with a region of collagen-like sequence followed by a C-type lectin domain. The polypeptides associate in threes to form a subunit containing a collagen-like helix, with three clustered lectin domains. These subunits associate into larger structures, usually with 12-18 polypeptides. The collectins bind to patterns of neutral sugars on surfaces (e.g. of micro-organisms) and mediate effector functions associated with killing/phagocytosis. MBL is the only collectin which activates complement. It resembles in quaternary structure the complement protein C1q, which recognizes targets via charge clusters. Binding of MBL to a surface activates MBL-associated serine proteases (MASPs) attached to MBL, and MASP-2 activates complement proteins C4 and C2. The MASPs are homologous to the C1q-associated proteases, C1r and C1s. MBL therefore activates complement by a mechanism very similar to C1q, and engages the opsonic activity of complement to clear micro-organisms. The serum concentration of MBL is very variable in humans. The variability is largely associated with mutations leading to amino acid substitutions in the collagen-like region which decrease MBL assembly and stability. Many studies demonstrate that MBL deficiency is associated with susceptibility to a range of infectious and inflammatory diseases.

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324 -332, 1 FAPGNYPAL 9 ) to promote its folding in vitro of H-2D b . We found that H-2D b can be stabilized by the pentapeptide 5 NYPAL 9 , which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D b molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove. Major histocompatibility complex (MHC)5 class I molecules have evolved to present peptide epitopes of 8 -10 amino acids to cytotoxic T cells. Many MHC class I⅐peptide structures have now been solved by x-ray crystallography, and they all have a common tertiary structure (1). The structure consists of a polymorphic heavy chain (HC) and a nonpolymorphic light chain ␤2-microglobulin (␤2-m), noncovalently associated to form a molecule with two membrane proximal Ig-like domains (the ␣3 and ␤2-m domains) that support a membrane distal ␣1-␣2 "superdomain." The peptide-binding site is formed by a deep cleft between two ␣-helices in this superdomain. Antigenic peptides are always bound in the same orientation, with their N and C termini lying buried deep in pockets that define the ends of the peptide-binding groove (the A and F pockets, respectively). In addition, so-called "anchor residues" make allele specific interactions with polymorphic class I residues located deep inside the binding groove, in "specificitydetermining pockets."The peptide⅐MHC class I complex is formed in the endoplasmic reticulum (ER) and marks the end point of antigen processing (2). During antigen processing, proteins are unfolded and partially hydrolyzed in the cytoplasm, and the resulting polypeptides (of between 8 and 40 amino acids) are translocated across the ER membrane by the transporter associated with antigen processing. Once in the ER, some long peptide epitope precursors can undergo further trimming by the aminopeptidase ERAAP (3, 4) and are selected for assembly with newly synthesized MHC class I molecules that is dependent on their interaction with cofactor molecules such as calreticulin, tapasin, and ERp57. The process results in the preferential release from the ER of class I molecules presenting peptides that bind stably. Recent evidence suggests that this selection of high affinity peptides in vivo may occur by a mechanism that is more complex and controlled than simple competition between potential ligands for binding to class I in the ER (2) and may involve editing of the MHC-bound peptide repertoire in the early secretory pathway of antigen-presenting cells.It is not known whether the loading or editing of class I MHC peptide cargo...

Class I major histocompatibility complexes (MHC) are heterotrimeric structures comprising heavy chains (HC), ␤ 2 -microglobulin (␤ 2 -m), and short antigenic peptides of 8 -10 amino acids. These components assemble in the endoplasmic reticulum and are released to the cell surface only when a peptide of the appropriate length and sequence is incorporated into the structure. The binding of ␤ 2 -m and peptide to HC is cooperative, and there is indirect evidence that the formation of a stable heterotrimer from an unstable HC:␤ 2 -m heterodimer involves a peptide-induced conformational change in the HC. Such a conformational change could ensure both a strong interaction between the three components and also signal the release of stably assembled class I MHC molecules from the endoplasmic reticulum. A peptide-induced conformational change in HC has been demonstrated in cell lysates lacking ␤ 2 -m to which synthetic peptides were added. Many features of this conformational change suggest that it may be physiologically relevant. In an attempt to study the peptide-induced conformational change in detail we have expressed a soluble, truncated form of the mouse H-2D b HC that contains only the peptide binding domains of the class I molecule. We have shown that this peptidebinding "platform" is relatively stable in physiological buffers and undergoes a conformational change that is detectable with antibodies, in response to synthetic peptides. We also show that the structural features of peptides that induce this conformational change in the platform are the same as those required to observe the conformational change in full-length HC. In this respect, therefore, the HC ␣ 1 and ␣ 2 domains, which together form the peptide binding site of class I MHC, are able to act independently of the rest of the molecule.

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IL-12 is secreted by macrophages and antigen presenting cells, and is a key cytokine at the interface between innate and acquired immunity, initiating cell-mediated (Thl -type) immune responses. We have previously shown using an ELISA method, that recombinant IL-12, both human and murine, binds specifically and with high affinity to heparin and heparan sulphate glycosaminoglycans (Hasan et al. J. Immunol. 1999Immunol. 162: 1064. IL-12 is a heterodimer, comprising 40 and 35kD subunits. We found that the p40 subunit in the absence of p35 also binds strongly to heparin. In order to characterise the interaction between IL-12 and glycosaminoglycans further, we have now found that N-desulphation, selective 6-0-desulphation, and selective 2-0-desulphation of heparin all result in preparations which show considerably reduced, but not abolished, binding. We therefore conclude that each of these three classes of sulphate plays some partial role in binding. We have also examined the ability of heparin and heparan sulphate to protect rIL-12 from proteolytic digestion in vitro. Both subunits are protected by heparin from degradation by the lysine-specific enzyme, Lys C, but the effect on the p40 subunit is particularly marked. The physiological importance of this protease protection now needs to be established. We are continuing to investigate the structural basis and functional consequences of the binding of IL-12 to heparin and heparan sulphate. 221 9 Regulation of human MBL-MASPs by CI-inhibitor and a2macroglobulin M.