Strong Cooperativity and Loose Geometry between CUB Domains Are the Basis for Procollagen C-Proteinase Enhancer Activity

Abstract

Procollagen C-proteinase enhancers (PCPE-1 and -2) specifically activate bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family during C-terminal processing of fibrillar collagen precursors. PCPEs consist of two CUB domains (CUB1 and CUB2) and one NTR domain separated by one short and one long linker. It was previously shown that PCPEs can strongly interact with procollagen molecules, but the exact mechanism by which they enhance BMP-1 activity remains largely unknown. Here, we used a series of deletion mutants of PCPE-1 and two chimeric constructs with repetitions of the same CUB domain to study the role of each domain and linker. Out of all the forms tested, only those containing both CUB1 and CUB2 were capable of enhancing BMP-1 activity and binding to a mini-procollagen substrate with nanomolar affinity. Both these properties were lost by individual CUB domains, which had dissociation constants at least three orders of magnitude higher. In addition, none of the constructs tested could inhibit PCPE activity, although CUB2CUB2NTR was found to modulate BMP-1 activity through direct complex formation with the enzyme, resulting in a decreased rate of substrate processing. Finally, increasing the length of the short linker between CUB1 and CUB2 was without detrimental effect on both activity and substrate binding. These data support the conclusion that CUB1 and CUB2 bind to the procollagen substrate in a cooperative manner, involving the short linker that provides a flexible tether linking the two binding regions.Tolloid proteinases have been shown to play important roles during embryogenesis and tissue remodeling. They control extracellular matrix synthesis as well as morphogenetic events such as dorso-ventral patterning, neural differentiation, and muscle growth (1). This control is achieved through proteolytic modifications of several matrix components (fibrillar and nonfibrillar procollagens, small leucine-rich proteoglycans, laminin 332, perlecan, and others), enzymes (lysyl oxidases) and growth factors or associated molecules (chordin, latent transforming growth factor-␤-binding protein-1, growth differentiation factors 8 and 11, prolactin, and others). In mammals, the tolloid family includes bone morphogenetic protein-1 (BMP-1), 2 mammalian tolloid, and mammalian tolloid like-1 and -2 (2). BMP-1 and mammalian tolloid are also known as "procollagen C-proteinases" (PCPs), because one of their key functions is to trigger collagen fibrillogenesis through cleavage of the C-terminal propeptides in fibrillar procollagens (3). Collagen fibrils then provide a scaffold for further deposition of other matrix molecules.Tolloid enzymes are assisted during collagen maturation by the procollagen C-proteinase enhancers-1 and -2 (PCPE-1 and -2), which can increase tolloid activity on the major fibrillar procollagens by Ͼ10-fold (4, 5) while not affecting the cleavage of other known tolloid substrates (6). PCPEs are rather small extracellular glycoproteins (ϳ50 kDa) consisting of, from the N ...

Procollagen C-proteinase enhancer grasps the stalk of the C-propeptide trimer to boost collagen precursor maturation

“…S2A) that CUB1-CUB2 produced in the baculovirus system had a similar circular dichroism (CD) spectrum to CUB1CUB2 obtained by trypsin digestion of full length PCPE-1 (nonmutated at the N-glycosylation site) expressed in human embryonic kidney 293 EBNA cells (17). In addition, we found both forms of CUB1CUB2 to be equally effective in enhancing BMP-1 activity on a miniprocollagen substrate, with a similar binding affinity as determined by Biacore (Fig.…”

“…More recently, cardiac fibrosis after chronic pressure overload was found to be diminished in mice lacking PCPE-2 (16). Although the precise mechanism of enhancement of proteinase activity remains to be elucidated, it is clear that this requires binding of PCPEs, or more precisely their CUB (Complement C1r/C1s, Uegf, BMP-1) domain region, to the procollagen C-propeptide trimer (17,18). Interestingly, the stoichiometry of binding is one molecule of PCPE to one molecule of procollagen (19), suggesting that the PCPE binding site on the C-propeptide trimer involves more than one polypeptide chain.…”

Metalloproteinases in Drosophila to Humans That Are Central Players in Developmental Processes

“…The CUB domains of PCPE1 bind procollagen (82) in a cooperative manner (83), and its NTR domain can bind BMP1 and mTLL1 (84,85), suggesting that PCPE1 may act as a linker that enhances procollagen-B/TP interactions. Moreover, enhancement of pCP activity by PCPE1 is potentiated by heparin or heparan sulfate, both of which bind the PCPE1 NTR domain, procollagen, and BMP1 (85,86), suggesting that heparan sulfate proteoglycans (HSPGs) may foster procollagen processing in vivo by bolstering formation of PCPE-procollagen-B/TP complexes (85,86).…”

Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate

“…The PCPE-1 molecule has been shown to be a rod-like molecule, with a length of ϳ15 nm (23). The short linker between the CUB domains of PCPE-1 provides a flexible tether linking two binding sites within the individual CUB domains that act cooperatively in the binding of PCPE-1 to the procollagen substrate (20).…”

“…The NTR domain of PCPE-1 also binds to ␤2-microglobulin, which may help initiate ␤2-microglobulin amyloid fibril formation in connective tissues (19). The CUB and NTR domains in PCPE-1 are separated by two linkers: a short linker (9 amino acids in human PCPE-1) between the two CUB domains and a long linker (44 amino acids in human PCPE-1) that is sensitive to proteolysis between the second CUB and the NTR domain (6,19,20). The NTR domain has homology with the N-terminal domain of tissue inhibitors of metalloproteinases and with the C-terminal domain of netrins, complement components C3, C4, and C5, and secreted frizzled-related proteins (16).…”