Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.
The murine BP-1 antigen (also called 6C3) is a homodimeric, phosphorylated cell surface glycoprotein that is expressed on immature B-lineage cells, bone marrow stromal cell lines, thymic cortical epithelial cells, endothelial cells, enterocytes, and renal proximal tubular cells. The amino acid sequence deduced from a BP-1 cDNA predicted a type II integral membrane protein with a zinc-binding motif (His-Glu-Xaa-Xaa-His) found in zinc-dependent metallopeptidases, and functional analysis suggested that BP-1 is aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7]. Here we constructed an expression vector in which the BP-1 cDNA was placed downstream from the SR alpha promoter and used this construct to transfect COS-7 and Ltk- cells. Both transfectants expressed the BP-1 antigen on the cell surface and APA activity. The enzymatic activity of recombinant APA was increased by Ca2+ and inhibited by Zn2+, consistent with previous reports with purified APA. Point mutation of one of the histidine residues in the zinc-binding motif to phenylalanine completely abolished APA enzymatic activity, suggesting the structure of the zinc-binding motif of APA is critical for catalytic activity. Both wild-type and mutant BP-1 were glycosylated, transported to the cell surface, and possessed molecular weights similar to native BP-1 molecules on the murine 18.81 pre-B-cell line. The successful expression of both wild-type and mutant APA should allow more precise analysis of the diverse physiological roles of this ectoenzyme.
Wang
J
J
Howard Hughes Medical Institute, University of Alabama, Birmingham 35294.
Cooper
M D
MD
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D043384:Glutamyl Aminopeptidase; D006639:Histidine; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D014162:Transfection; D015032:Zinc / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006639:Histidine; D004247:DNA; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; C062906:BP-1-6C3 antigen; D008666:Metalloendopeptidases; D015032:Zinc