Identification of Mouse CPX-2, a Novel Member of the Metallocarboxypeptidase Gene Family: cDNA Cloning, mRNA Distribution, and Protein Expression and Characterization

Abstract

A novel member of the metallocarboxypeptidase gene family was identified from its homology with carboxypeptidase E and has been designated CPX-2. The cDNA of 2500 nucleotides encodes a protein of 764 amino acids that contains an N-terminal signal peptide-like sequence, a 158-residue discoidin domain, and a 400-residue carboxypeptidase domain. The 400-residue metallocarboxypeptidase domain has 59% amino acid identity with a protein designated AEBP-1; 44% to 46% identity with carboxypeptidases E, N, and Z; and lower homology with other members of the metallocarboxypeptidase gene family. The discoidin domain of CPX-2 has 22% amino acid identity with the carbohydrate-binding domain of discoideum-I, 29% to 34% identity with the phospholipid-binding domain of human factors V and VIII, and 59% identity with the discoidin-like domain on AEBP-1. CPX-2 is missing several of the predicted active-site residues that are conserved in most other members of the metallocarboxypeptidase gene family and which are thought to be required for enzyme activity. Expression of CPX-2 using the baculovirus system produced several forms of protein, from 80 to 105 kDa, but no detectable activity toward a variety of carboxypeptidase substrates. A shorter 50-kDa form of CPX-2, which contains the carboxypeptidase domain but not the discoidin domain, was also inactive when expressed in the baculovirus system. CPX-2 is able to bind to Sepharose-Arg; this binding is blocked by 10 mM Arg. Northern blot analysis showed CPX-2 mRNA in mouse brain, liver, kidney, and lung. In situ hybridization analysis of brain revealed a broad distribution. Areas that are enriched in CPX-2 include the hippocampus, cerebral cortex, median eminence, and choroid plexus. Taken together, these data suggest a widespread function for CPX-2, possibly as a binding protein rather than an active carboxypeptidase.

Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

“…This third domain is predicted to be inactive due to the naturally occurring loss of the equivalent active site Glu. Other members of the metallocarboxypeptidase gene family that lack a corresponding Glu (CPX2 and AEBP1) are also inactive toward standard CPE substrates (32,34), thus leading to the proposal that these family members are binding proteins rather than active enzymes. It is possible that the third domain of CPD also functions as a binding protein; from the present study, this third domain is not a functional carboxypeptidase.…”

“…However, low levels of mature peptides are detected, indicating that another enzyme is able to partially compensate for the absence of CPE activity. A search for novel CPE-like enzymes led to the identification of CPD, carboxypeptidase Z, and proteins designated CPX-1 and CPX-2 (27,(32)(33)(34). In addition, another group identified a protein designated AEBP1 as a novel member of the carboxypeptidase gene family (35).…”

“…In addition, another group identified a protein designated AEBP1 as a novel member of the carboxypeptidase gene family (35). Of these proteins, only CPD and carboxypeptidase Z demonstrate activity toward standard CPE substrates (27,(32)(33)(34). The cellular distribution of carboxypeptidase Z is restricted to specific cell types, such as the leptomeningeal cells in brain (36).…”

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

“…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].…”

Glu300 of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway

“…These family members are all enzymatically active, and are 30 -40-kDa proteins. The other group includes CPE, carboxypeptidase M (CPM), carboxypeptidase N (CPN), carboxypeptidase D (CPD), carboxypeptidase Z (CPZ), and three proteins designated CPX1, CPX2, and AEBP1 (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Except for CPD, which is 180 kDa and contains 3 distinct carboxypeptidase-like domains, the members of this family contain a single carboxypeptidase domain of approximately 400 amino acids.…”