Identification of Mouse CPX-1, a Novel Member of the Metallocarboxypeptidase Gene Family with Highest Similarity to CPX-2

Lei, Yinghong; Xin, Xiaonan; Morgan, Daniel J.; Pintar, John E.; Fricker, Lloyd D.

doi:10.1089/104454999315565

Cited by 55 publications

(57 citation statements)

References 50 publications

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“…Empirical evidence suggests it lacks catalytic activity [3] and this finding is consistent with sequence alignments which reveal substitution of two residues critical for substrate binding (R117 and Y248 in CPB are replaced by Val and His residues, V408 and H558, in CPX-1) [2,3]. Thus, the molecular role and biological function of CPX-1 are unclear.…”

Section: Introductionsupporting

confidence: 65%

“…CPX-1 was first identified and characterised almost 20 years ago when it was shown to be a secreted protein subject to N-glycosylation [3]. Empirical evidence suggests it lacks catalytic activity [3] and this finding is consistent with sequence alignments which reveal substitution of two residues critical for substrate binding (R117 and Y248 in CPB are replaced by Val and His residues, V408 and H558, in CPX-1) [2,3].…”

Section: Introductionsupporting

confidence: 55%

“…At the molecular level CPX-1 is a modular protein containing a classic signal peptide, a DSD and a catalytically inactive CP domain [3] (see Fig 1A). Given the absence of enzymatic activity and the recognised functional and biological importance of the DSDs in a range of other proteins [6,7,8] we reasoned that increased understanding of the putative role of the DSD in CPX-1 would help direct future studies investigating the biological function of CPX-1.…”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

“…CPX-1 has been reported to be secreted in an N-glycosylated form [3]. In silico analysis revealed 4 putative N-glycosylation sites in human CPX-1 including one situated within the DSD (Fig 1A).…”

Section: Cpx-1 Is Secreted In An N-glycosylation Dependent Mannermentioning

confidence: 99%

“…The three members that lack catalytic activity, CPX-1, CPX-2 and aortic carboxypeptidase-like protein/adipocyte enhancer binding protein 1 (ACLP/AEBP1) form a discrete subset [2]. Their CP domains show the highest degree of homology within the family with each lacking one or more residues critical for enzymatic activity and/or substrate binding [2,3,4]. Furthermore, each contains an N-terminal signal peptide followed by a discoidin domain (DSD), a domain not present in the other family members [2].…”

Section: Introductionmentioning

confidence: 99%

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

Kim

O’Neill

Whitehead

2015

Biochemical and Biophysical Research Communications

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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.

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Section: Introductionsupporting

confidence: 65%

Section: Introductionsupporting

confidence: 55%

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

Section: Cpx-1 Is Secreted In An N-glycosylation Dependent Mannermentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Kim

O’Neill

Whitehead

2015

Biochemical and Biophysical Research Communications

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Metallocarboxypeptidases

Vendrell¹,

Avilés²,

Fricker³

2004

Handbook of Metalloproteins

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Metallocarboxypeptidases (CP) catalyze the removal of C‐terminal amino acids from proteins and/or peptides. The different members of the CP family differ in their specificity for C‐terminal residues and physiological function and can be divided into two subfamilies. Members of the A/B subfamily are generally produced as proenzymes, contain an approximately 300‐residue CP catalytic domain, have greatest amino acid sequence identity to the exocrine pancreatic enzymes CPA and CPB, and prefer hydrophobic or basic residues. They function in the breakdown of peptides in food or in other physiological processes ranging from inflammation to fibrinolysis. Members of the N/E group cleave C‐terminal basic residues and are not produced as inactive proenzymes but contain an approximately 80‐residue region following the 300‐residue CP domain with structural homology to transthyretin. They act either extra‐ or intracellularly in the processing of peptide hormones and neurotransmitters and other physiologically relevant peptides. The tertiary folding of CPs corresponds to the α/β hydrolase fold and is formed by a central mixed parallel/antiparallel eight‐strand β‐sheet, with a 120° twist between the first and the last strand, over which eight α‐helices pack on both sides to form a globular molecule. All of the enzymatically active CPs bind one atom of Zn 2+ at the active site. Some members of the CP family that are inactive against standard CP substrates lack some of the cation‐binding ligands and may therefore not be able to bind the metal.

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Carboxypeptidases

Avil��s¹,

Vendrell²

2002

Wiley Encyclopedia of Molecular Medicine

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Identification of Mouse CPX-1, a Novel Member of the Metallocarboxypeptidase Gene Family with Highest Similarity to CPX-2

Cited by 55 publications

References 50 publications

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

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

Metallocarboxypeptidases

Carboxypeptidases

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