Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Biochem Biophys Res Commun. 2015 Dec 25;468(4):894-9. doi: 10.1016/j.bbrc.2015.11.053. Epub 2015 Nov 19.

Abstract

Carboxypeptidase X-1 (CPX-1) is an atypical member of the carboxypeptidase (CP) family of proteins involved in a variety of physiological and pathological processes. However, unlike most other family members CPX-1 lacks catalytic activity making its biological function unclear. CPX-1 contains a 160 amino acid discoidin domain (DSD) that serves as a binding domain in other proteins prompting us to investigate a putative functional role for this domain in CPX-1. Sequence alignment confirmed the overarching homology between the DSD of CPX-1 and other DSDs whilst more detailed analysis revealed conservation of the residues known to form the collagen-binding trench within the DSD of the discoidin domain receptors (DDRs) 1 and 2. Biochemical characterisation of transiently expressed human CPX-1 revealed that CPX-1 was secreted in an N-glycosylation-dependent manner as treatment with the N-glycosylation inhibitor tunicamycin inhibited secretion concomitant with a reduction in CPX-1 mobility on Western blot. Using a collagen pull-down assay we found that secreted CPX-1 bound collagen and this appeared independent of N-glycosylation as treatment with PNGaseF did not affect binding. Further analysis under non-reducing and reducing (+DTT) conditions revealed that CPX-1 was secreted in both monomeric and dimeric forms and only the former bound collagen. Finally, mutation of a key residue situated within the putative collagen-binding trench within the DSD of CPX-1 (R192A) significantly reduced secretion and collagen-binding by 40% and 60%, respectively. Collectively these results demonstrate that CPX-1 is a secreted collagen-binding glycoprotein and provide a foundation for future studies investigating the function of CPX-1.

Keywords: Carboxypeptidase; Collagen; Discoidin domain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Collagen / chemistry*
  • Collagen / metabolism*
  • Cricetulus
  • Enzyme Activation
  • Glycoproteins / chemistry*
  • Glycoproteins / metabolism*
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Metalloexopeptidases / chemistry*
  • Metalloexopeptidases / metabolism*
  • Protein Binding
  • Protein Structure, Tertiary
  • Substrate Specificity

Substances

  • CPXM1 protein, human
  • Glycoproteins
  • Collagen
  • Metalloexopeptidases
  • Cpxm1 protein, mouse