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

Abstract

Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.

Disruption of the murine procollagen C-proteinase enhancer 2 gene causes accumulation of pro-apoA-I and increased HDL levels

“…PCPE2, like PCPE1, consists of two cubulin (CUB) domains (CUB1 and CUB2) involved in protein-protein interaction. While several studies have been published addressing the physiological role of PCPE1 ( 13,14 ), the function of PCPE2 remains unclear. PCPE2-defi cient (PCPE2 KO) mice are viable and fertile, and they do not show gross developmental abnormalities ( 15 ).…”

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

“…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). HSPGs may also bind PCPEs to cell surfaces (86). PCPE1 enhancement of B/TPs seems specific to pCP activity, as PCPE1 failed to enhance cleavage of a number of other substrates in vitro (87).…”

“…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). HSPGs may also bind PCPEs to cell surfaces (86).…”

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake

“…3, A-D). PCPE2 enhances procollagen processing and is abundantly expressed in the heart, aorta, adipose tissue, and trabecular meshwork (27,29,38). The expression of PCPE1, which is related to PCPE2, was not increased as a result of PCPE2 deletion (Fig.…”

“…The tandem CUB domains are followed by a C-terminal netrin-like (NTR) domain (27,29). CUB domains are commonly found in extracellular membrane proteins that mediate protein-protein interactions (33)(34)(35)(36), whereas NTR domains bind to cell surface glycosaminoglycans (37,38), presumably anchoring PCPE2 to the extracellular matrix. Interestingly, BMP1, in addition to catalyzing procollagen processing, removes the apoA-I six-amino acid propeptide (39).…”