Substrate-specific Modulation of a Multisubstrate Proteinase

Abstract

Members of the bone morphogenetic protein-1/tolloid (BMP-1/Tld) family of metalloproteinases, also known as procollagen C-proteinases (PCPs), control multiple biological events (including matrix assembly, cross-linking, cell adhesion/migration and pattern formation) through enzymatic processing of several extracellular substrates. PCP activities on fibrillar procollagens can be stimulated by another family of extracellular proteins, PCP enhancers (PCPE-1, PCPE-2), which lack intrinsic enzymatic activity. While PCPs have multiple substrates, the extent to which PCPEs is involved in the processing of proteins other than fibrillar procollagens is unknown. In the experiments reported here, PCPE-1 was found to have no effect on the in vitro BMP-1 processing of procollagen VII, the procollagen V N-propeptide, the laminin 5 ␥2 chain, osteoglycin, prolysyl oxidase, or chordin. In contrast, PCPE-1 enhanced C-terminal processing of human fibrillar procollagen III but only when this substrate was in its native, disulfidebonded conformation. Surprisingly, processing of procollagen III continued to be enhanced when essentially all the triple-helical region was removed. These and previous results (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. In recent years, it has become clear that members of the tolloid family of metalloproteinases (BMP-1, 1 mTld, mTLL-1, mTLL-2) are key players in morphogenesis through their ability to control and synchronize the processing of multiple extracellular substrates (1-25). Also called procollagen C-proteinases, tolloid proteinases trigger collagen fibril formation in the extracellular matrix (26) by cleaving the C-propeptide regions from the major fibrillar procollagens (I, II, and III). They are also involved in precursor processing of the minor fibrillar collagens V and XI, necessary for the regulation of heterotypic fibril assembly, in both the N-and C-propeptide regions (8,15,17,19,23,27). Tolloid proteinases also cleave precursor forms of small leucine-rich proteoglycans such as biglycan (13) and osteoglycin (24). While some of these proteoglycans have been shown to modulate the kinetics of assembly as well as fibril diameter (28), functional differences between mature and precursor forms are not yet clearly established (24). Biglycan is also important for bone formation (29) and more recently, dentin matrix protein-1, a protein involved in mineralization, has been found to be a substrate for tolloid proteinases (22).Another important function early attributed to tolloid proteinases is the maturation of the precursor form of lysyl oxidase (LOX) (5, 30), the enzyme responsible for the stabilization of collagen and elastin networks through the formation of covalent cross-links. This tolloid-dependent processing has recently gained in significance with the demonstration that the cleaved propeptide of proLOX accounts for the ras-recision gene activity of this protein (31). In addition, ...

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold

“…Overlapping oligonucleotides (Table 1) were purchased from MWG-BIOTECH (Courtaboeuf, France). The mutated inserts were then subcloned into pCEP4 (Invitrogen) using the KpnI and BamHI sites for semi-stable transfections into 293-EBNA cells (28). The sequences of all mutants were confirmed by double-stranded DNA sequencing (Genome Express, France).…”

“…Recombinant Proteins-The BMP-1 FLAG construct was obtained by PCR using the BMP-1 cDNA inserted in pCEP4 as a template (28). The FLAG sequence was inserted immediately contiguous to the BMP-1 sequence before the STOP codon.…”

“…BIAcore studies have shown that PCPE-1 binds to both the C-propeptide region as well as elsewhere in the procollagen molecule (35). Recent studies using a miniprocollagen III substrate have shown that enhancing activity is unaffected by removal of essentially all of the triple-helical region using highly purified bacterial collagenase (28). Taken together, these data suggest that PCPE-1 binds to the non-triple-helical telopeptide region N-terminal to the BMP-1 cleavage site, in addition to the C-propeptide region, thereby inducing a conformational change that facilitates the action of BMP-1.…”

“…During developmental patterning, for example, it has recently been shown that the frizzled-related protein sizzled is an endogenous inhibitor of BMP-1 (3,26), whereas complex formation involving the protein-twisted gastrulation stimulates the activity of BMP-1 on chordin and exposes a new cleavage site (27). Procollagen C-proteinase enhancers (PCPE-1 and -2) also stimulate the activities of tolloid proteinases, in a substrate-specific manner (28). PCPE-1 has no effect on cleavage of several BMP-1 substrates (28), including chordin (29), prolysyl oxidase, osteoglycin, the laminin 5 ␥2 chain, procollagen VII, and the N-propeptide region of procollagen V. In contrast, both PCPE-1 and -2 have been shown to stimulate the activities of tolloid proteinases during cleavage of the C-propeptide regions of the major fibrillar procollagens (types I, II, and III) (28,30,31).…”

“…Procollagen C-proteinase enhancers (PCPE-1 and -2) also stimulate the activities of tolloid proteinases, in a substrate-specific manner (28). PCPE-1 has no effect on cleavage of several BMP-1 substrates (28), including chordin (29), prolysyl oxidase, osteoglycin, the laminin 5 ␥2 chain, procollagen VII, and the N-propeptide region of procollagen V. In contrast, both PCPE-1 and -2 have been shown to stimulate the activities of tolloid proteinases during cleavage of the C-propeptide regions of the major fibrillar procollagens (types I, II, and III) (28,30,31). Targeted deletion of the PCPE-1 gene has recently been shown to lead to aberrant collagen fibril formation and impaired biomechanical properties of bone tissue (32).…”