Role of the occluding loop in cathepsin B activity.

Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft. Variants of human procathepsin B, where all or part of this element was deleted, were expressed in the yeast Pichia pastoris. A mutant, where the 12 central residues of the occluding loop were deleted, autoprocessed, albeit more slowly than the wild type proenzyme, to yield a mature form of the enzyme with endopeptidase activity comparable with the wild-type cathepsin B, but totally lacking exopeptidase activity. This deletion mutant showed a 40-fold higher affinity for the inhibitor cystatin C, suggesting that the occluding loop normally restricts access of this inhibitor to the active site. In addition, the binding affinity of the cathepsin B propeptide, which is a potent inhibitor of this enzyme, was 50-fold increased, consistent with the finding that the loop reorients on activation of the proenzyme. These results suggest that the endopeptidase activity of cathepsin B is an evolutionary remnant since, as a consequence of its membership in the papain family, the propeptide must be able to bind unobstructed through the full length of the active site cleft.

Activation of human complement serine-proteinase C1r is down-regulated by a Ca(2+)-dependent intramolecular control that is released in the C1 complex through a signal transmitted by C1q.

The activation of human C1, a Ca(2+)-dependent complex proteinase comprising a non-enzymic protein, C1q, and two serine proteinases, C1r and C1s, is based primarily on the intrinsic property of C1r to autoactivate. The aim of the present study was to investigate the mechanisms involved in the regulation of C1r autoactivation, with particular attention to the role of Ca2+ ions. Spontaneous activation of proenzyme C1r was observed upon incubation in the presence of EDTA, whereas Ca2+ ions reduced markedly the activation process. Several lines of evidence indicated that Ca2+ inhibited the intramolecular activation reaction but had little or no effect on the intermolecular activation reaction. C1q caused partial release of this inhibitory effect of Ca2+. Complete stabilization of C1r in its proenzyme form was obtained upon incorporation within the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer, and a comparable effect was observed when C1s was replaced by its Ca(2+)-binding alpha-fragment. Both tetramers, C1s-C1r-C1r-C1s and C1s alpha-C1r-C1r-C1s alpha, readily associated with C1q to form 16.0 S and 14.7 S complexes respectively in which C1r fully recovered its activation potential. Both complexes showed indistinguishable activation kinetics, indicating that the gamma B catalytic region of C1s plays no role in the mechanism that triggers C1r activation in C1. The collagen-like fragments of C1q retained the ability to bind to C1s-C1r-C1r-C1s, but, in contrast with intact C1q, failed to induce C1r activation in the resulting complex at temperatures above 25 degrees C. On the basis of these observations it is proposed that activation of the serine-proteinase domain of C1r is controlled by a Ca(2+)-dependent intramolecular mechanism involving the Ca(2+)-binding alpha-region, and that this control is released in C1 by a signal originating in C1q and transmitted through the C1q/C1r interface.

Assembly of the C1 complex.

The C1 complex of complement is a Ca(2+)-dependent complex protease comprising two loosely interacting subunits. C1q, the recognition subunit, is an hexameric protein with six peripheral globular domains, each connected through collagen-like "arms" to a central fibril-like "stalk". The catalytic subunit, C1s-C1r-C1r-C1s, is a Ca(2+)-dependent tetrameric association of two serine protease zymogens, C1r and C1s, that are sequentially activated by cleavage of a single peptide bond, upon binding of C1 to activators. Each monomeric protease is comprised of six structural motifs which form at least four domains, distributed in two functionally distinct regions, alpha (N-terminal) and gamma-B (C-terminal). The catalytic (gamma-B) regions of C1r and C1s are respectively located in the centre and at each end of the isolated tetramer, and the Ca(2+)-dependent C1r-C1s associations are mediated by the interaction (alpha) regions, which contain one Ca2+ binding site each. Physicochemical and electron microscopy studies indicate that the tetramer, which is highly elongated, folds into a more compact conformation upon interaction with C1q. Various models for C1 have been proposed, in which the tetramer either interacts with the outside part of the C1q arms (O- and W-shaped models), or is folded within the C1q arms (S- or 8-shaped models). These models are discussed in light of available information and in consideration of the structural requirements of C1 activation and function.

Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement.

The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5'-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[-3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites.

Effect of lactoperoxidase-catalyzed iodination on the Ca(2+)-dependent interactions of human C1s. Location of the iodination sites.

C-1s, one of the two serine proteases of C-1, the first component of complement, has the ability to mediate heterologous (C-1r-C-1s) as well as homologous (C-1s-C-1s) Ca(2+)-dependent interactions both involving the NH2-terminal alpha region of its A chain. Lactoperoxidase-catalyzed iodination of C-1s in its monomeric form was found to abolish its ability to form Ca(2+)-dependent homodimers, without impairing its ability to mediate C-1r-C-1s heteroassociation. C-1s iodinated in its dimeric form, in contrast, fully retained the ability to self-associate. With a view to identify the tyrosine residues iodinated in each case, C-1s was radioiodinated in its monomeric and dimeric forms, and comparative tryptic mapping was performed on the resulting 125I-labeled A chains. Most of the tyrosine residues either were not iodinated or were equivalently but not in the dimer. Conversely, Tyr-52 and Tyr-147 were iodinated only in the dimer. These results provide further evidence that the structural determinants of C-1s required for Ca2+ binding and Ca(2+)-dependent protein-protein interactions are contributed by both the NH2-terminal motif I (positions 1-110) and the epidermal growth factor like motif II (positions 111-159) of the alpha region. On the basis of available information, tentative models of the C-1s-C-1s and C-1r-C-1s Ca(2+)-dependent interactions are proposed.