title: Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein.
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.
authors.affiliation: Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia.
authors.forename: Yu-Hee
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia.
authors.forename: Hayley M
authors.initials: HM
authors.lastname: O'Neill
authors.affiliation: Mater Research Institute-University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia. Electronic address: jon.whitehead@mater.uq.edu.au.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Whitehead
mesh_terms: D000818:Animals; D016466:CHO Cells; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism*; D003412:Cricetulus; D004789:Enzyme Activation; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D045727:Metalloexopeptidases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: C000621999:CPXM1 protein, human; D006023:Glycoproteins; D003094:Collagen; D045727:Metalloexopeptidases; C117650:Cpxm1 protein, mouse
keywords: Carboxypeptidase; Collagen; Discoidin domain