Efrat Kessler

Bone morphogenetic protein-1 : the type I procollagen C-proteinase

Bone morphogenetic proteins (BMPs) are bone-derived factors capable of inducing ectopic bone formation. Unlike other BMPs, BMP-1 is not like transforming growth factor-β (TGF-β), but it is the prototype of a family of putative proteases implicated in pattern formation during development in diverse organisms. Although some members of this group, such as Drosophila tolloid (TLD), are postulated to activate TGF-β-like proteins, actual substrates are unknown. Procollagen C-proteinase (PCP) cleaves the COOH-propeptides of procollagens I, II, and III to yield the major fibrous components of vertebrate extracellular matrix. Here it is shown that BMP-1 and PCP are identical. This demonstration of enzymatic activity for a BMP-1/TLD-like protein links an enzyme involved in matrix deposition to genes involved in pattern formation.

COL1 C-propeptide cleavage site mutations cause high bone mass osteogenesis imperfecta

Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C‐propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1–L4 DXA Z‐score was +3.9 and pQCT vBMD was+3.1; Patient 2 had L1–L4 DXA Z‐score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC‐collagen into matrix leads to increased bone mineralization. FTIR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C‐propeptide cleavage is crucial to normal bone mineralization. Hum Mutat 32:1–12, 2011.

Evidence for a Protein that Enhances the Activity of Type I Procollagen C-Proteinase

Gel-filtration separated type I procollagen C-proteinase from a glycoprotein that enhanced the enzyme activity by approximately 4-fold. The enhancer was purified by affinity chromatography on a column of Sepharose coupled to the carboxyl propeptide of type I procollagen. Sodium-dodecyl-sulfate- polyacrylamide gel electrophoresis of the affinity-purified enhancer revealed two active major protein bands with molecular weights of 36 and 34 kdal. Both proteins were glycosylated, as shown by binding to concanavalin-A. The enhancer is extremely heat stable (100 degrees C, 15 min) but its activity is totally abolished by treatment with trypsin or bacterial elastase. The enhancer does not alter the digestion intermediates or final products of the enzymatic reaction but it changes the kinetic properties of the reaction, increasing the apparent Km and Vmax values 16- and 20-fold, respectively. It is suggested that the enhancer might play a regulatory role in procollagen processing.

Partial Purification and Characterization of a Procollagen C-Proteinase from the Culture Medium of Mouse Fibroblasts

A procollagen C-proteinase was purified about 100-fold from the medium of cultured mouse fibroblasts by a combination of ammonium sulfate precipitation, gel-filtration, and affinity chromatography on a column of Sepharose coupled to the carboxyl propeptide of type I procollagen. The purified enzyme did not exhibit other proteolytic activities, and it cleaved type I, II and III procollagens to produce the corresponding pN alpha chains and carboxyl propeptides as the only products. Amino acid sequencing of the first 14-18 residues at the N-terminus of the carboxyl propeptides generated by the enzyme from human pro alpha 1(I), pro alpha 2(I) and pro alpha 1(III) chains showed that the cleavage occurred at the physiological site, i.e. at the specific Ala-Asp bond in the pro alpha 1(I) and pro alpha 2(I) chains, and at the specific Gly-Asp bond in the pro alpha 1(III) chain. The pH optimum of the enzyme is 8.5 and its molecular weight as estimated by gel-filtration is about 125,000 daltons. The enzyme is inhibited by metal-chelators, various amines, dithiothreitol, N-ethylmaleimide and serum, but it is insensitive to pepstatin, leupeptin and serine proteases inhibitors. The enzyme differs from the C-proteinase described by Njieha et al. (Biochemistry 21:757-764, 1982), and the catheptic activities reported by Davidson et al. (Eur. J. Biochem 100:551-558, 1979) and Helseth and Veis (Proc. Natl. Acad. Sci. USA 81:3302-3306, 1984). The specificity of the enzyme is offered as evidence for a unique, C-proteinase, and its recovery from culture medium supports an extracellular location for procollagen processing.

The CUB domains of procollagen C-proteinase enhancer control collagen assembly solely by their effect on procollagen C-proteinase/bone morphogenetic protein-1

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

Expression of Procollagen C-proteinase enhancer in cultured rat heart fibroblasts: Evidence for co-regulation with type I collagen

Procollagen processing by procollagen C‐proteinase (PCP) is an important step in collagen deposition. This reaction is stimulated by another glycoprotein, known as PCP enhancer. The objective of this study was to identify factors that regulate the expression of PCP enhancer in cardiac fibroblasts and examine possible correlation with collagen expression. Rat heart fibroblasts were cultured in the presence or absence of three known stimulators of collagen synthesis: ascorbic acid, TGF‐β, and aldosterone. The mRNA and protein levels of PCP enhancer and collagen type I were each assessed using Northern and Western blotting, respectively. Expression of PCP was assessed by RT‐PCR and its activity in the culture media was determined using radioactive procollagen as the substrate. The levels of PCP enhancer mRNA increased 1.5‐ to 2‐fold in response to ascorbate, TGF‐β, or aldosterone. This increase was paralleled by an up to fourfold increase in the level of the pro α1(I) collagen chain transcript and was accompanied by a marked increase in the levels of the respective proteins in the culture media. PCP activity in the culture media was also increased, apparently, without effect on its expression. These results indicate that expression of PCP enhancer in cultured rat heart fibroblasts is coordinated with that of collagen. The observed augmentation of PCP activity may be a consequence of the increase in the levels of PCP enhancer in the culture media. J. Cell. Biochem. 90: 397–407, 2003.

Identification of binding partners interacting with the α1-N-propeptide of type V collagen.

The predominant form of type V collagen is the [α1(V)]₂α2(V) heterotrimer. Mutations in COL5A1 or COL5A2, encoding respectively the α1(V)- and α2(V)-collagen chain, cause classic EDS (Ehlers-Danlos syndrome), a heritable connective tissue disorder, characterized by fragile hyperextensible skin and joint hypermobility. Approximately half of the classic EDS cases remain unexplained. Type V collagen controls collagen fibrillogenesis through its conserved α1(V)-N-propeptide domain. To gain an insight into the role of this domain, a yeast two-hybrid screen among proteins expressed in human dermal fibroblasts was performed utilizing the N-propeptide as a bait. We identified 12 interacting proteins, including extracellular matrix proteins and proteins involved in collagen biosynthesis. Eleven interactions were confirmed by surface plasmon resonance and/or co-immunoprecipitation: α1(I)- and α2(I)-collagen chains, α1(VI)-, α2(VI)- and α3(VI)-collagen chains, tenascin-C, fibronectin, PCPE-1 (procollagen C-proteinase enhancer-1), TIMP-1 (tissue inhibitor of metalloproteinases-1), MMP-2 (matrix metalloproteinase 2) and TGF-β1 (transforming growth factor β1). Solid-phase binding assays confirmed the involvement of the α1(V)-N-propeptide in the interaction between native type V collagen and type VI collagen, suggesting a bridging function of this protein complex in the cell-matrix environment. Enzymatic studies showed that processing of the α1(V)-N-propeptide by BMP-1 (bone morphogenetic protein 1)/procollagen C-proteinase is enhanced by PCPE-1. These interactions are likely to be involved in extracellular matrix homoeostasis and their disruption could explain the pathogenetic mechanism in unresolved classic EDS cases.

Procollagen type I C-proteinase enhancer is a naturally occurring connective tissue glycoprotein

Using antibodies to the procollagen C-proteinase enhancer of mouse fibroblast culture medium, we have screened by immunoblotting extracts of several post natal mouse and rat tissues for the presence of the enhancer antigen. All rodent connective tissues were relatively rich in enhancer; lower amounts were found in skeletal muscle and heart and essentially no enhancer was detected in kidney, liver or brain. The amounts of enhancer in mouse tendon and calvaria extracts were age related, with highest amounts in 11 and 19 d tendons and in 1 d calvaria-the times of rapid growth of these organs. The results suggest that procollagen C-proteinase enhancer is a specific connective tissue glycoprotein that is likely to regulate procollagen processing in vivo.

Molecular events that contribute to lysyl oxidase enzyme activity and insoluble collagen accumulation in osteosarcoma cell clones

Maximum collagen synthesis and maximum accumulation of insoluble collagen occur at different phenotypic stages in developing osteoblastic cell cultures. Insoluble collagen accumulation depends in part on the activity of extracellular enzymes including procollagen N‐proteinases, procollagen C‐proteinase (derived from the BMP1 gene), and lysyl oxidase. In addition to its action on procollagen, procollagen C‐proteinase processes prolysyl oxidase to mature 32‐kDa lysyl oxidase. The regulation of extracellular activities that control insoluble collagen accumulation has not been studied extensively. The present study compares molecular events that control production of a collagenous mineralized extracellular matrix in vitro among five different murine osteosarcoma cell clones derived from the same tumor, but which differ in their ability to produce an insoluble mineralized matrix. Levels of insoluble type I collagen, insoluble calcium, bone morphogenetic protein 1 (BMP‐1), and lysyl oxidase expression, lysyl oxidase biosynthesis, lysyl oxidase activity, and prolysyl oxidase processing activity were determined. Results surprisingly indicate that lysyl oxidase activity is not related closely to lysyl oxidase messenger RNA (mRNA) levels among the different cell clones. However, it appears that BMP‐1‐dependent prolysyl oxidase processing could contribute to the observed lysyl oxidase activity. Highest collagen and BMP‐1 mRNA levels, prolysyl oxidase processing activity, and lysyl oxidase activity occurred in a cell clone (K8) that showed the highest levels of insoluble collagen accumulation. Culture media from a cell clone (K37) that accumulates little insoluble collagen or calcium but expresses high levels of lysyl oxidase mRNA contained low molecular weight fragments of lysyl oxidase protein and showed low lysyl oxidase activity. By contrast the K14 cell line exhibits relatively high lysyl oxidase activity and collagen accumulation, but low levels of mature lysyl oxidase protein. Together, these studies indicate that catabolic as well as anabolic activities are important in regulating insoluble collagen accumulation in osteoblastic cells. In addition, results suggest that products of genes homologous to lysyl oxidase may contribute to observed lysyl oxidase activity.

The Collagen V Homotrimer [α1(V) ] 3 Production Is Unexpectedly Favored over the Heterotrimer [α1(V)]2α2(V) in Recombinant Expression Systems

Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [α1(V)]3 homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V α-chains showed that, contrary to fibroblasts, collagen V α-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly.