Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.

Abstract

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-GluXaa-Xaa-His) found in zinc-dependent metallopeptidases, and functional analysis suggested that BP-1 is aminopeptidase A [APA; L-a-aspartyl(L-ar-glutamyl)-peptide hydrolase, EC 3.4.11.71. Here we constructed an expression vector in which the BP-1 cDNA was placed downstream from the SRa promoter and used this construct to transfect COS-7 and Ltkcells. 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.The differentiation of B cells requires interaction of lymphocyte precursors with other cell types in the microenvironments of fetal liver and adult bone marrow. Cell surface proteins expressed by developing B-lymphocyte precursors are thought to be involved in this highly complex, regulated process. The BP-1 antigen was identified as a homodimeric, phosphorylated cell surface glycoprotein on pre-B-cells and newly formed B cells in mouse bone marrow (1-3). This molecule, independently identified as a pre-B-cell neoplasiaassociated antigen and named 6C3 in another laboratory (4), is also expressed on certain bone marrow stromal cell lines and a subpopulation of thymic cortical epithelial cells (5-7).In mice, expression of the BP-1 (6C3) antigen on bone marrow-derived stromal cell lines correlates with their ability to support pre-B-cell growth (5). Interleukin 7 (IL-7) preferentially induces proliferation of bone marrow-derived pre-Bcells and up-regulates their expression of the BP-1 antigen (7,8), whereas its expression is extinguished in mature B lymphocytes and plasma cells (1). All of these observations suggest that the BP-1 molecule may play a crucial role in regulating growth and differentiation of early B-lineage cells.The BP-1 cDNA has been cloned recently (9), and the deduced amino acid sequence predicts a type II integral membrane protein with a zinc-binding motif-uncharged residue (Xaa)-Xaa-His-Glu-Xaa-Xaa-His-Xaa-hydrophobic residue-that is conserved among members of the zincd...

Migration‐stimulating factor displays HEXXH‐dependent catalytic activity important for promoting tumor cell migration

“…This type of mutation is known to abolish the catalytic activity of zinc-dependent metalloenzymes. 23,24 These results demonstrate that the structure of the HEXXH motif is critical for Fn-proteinase activity and suggest that this sequence is the functional zinc-binding site of Fn-proteinase.…”

The C-terminal domain, but not the interchain disulphide, is required for the activity and intracellular trafficking of aminopeptidase A

“…On expression in COS-1 cells the resulting mutant protein, G594∆, was readily detected with a polyclonal antibody raised against murine APA [23]. Despite the fact that all the residues currently identified as critical for metal binding and catalysis in APA are located in the 107 kDa domain [14][15][16][17][18], and thus are present in the G594∆ mutant, no enzymic activity was associated with the recombinant protein. This would suggest that either there is one or more critical catalytic residues within the Cterminal 45 kDa domain or that the C-terminal domain is required for correct folding of the remainder of the protein.…”

“…cDNAs encoding the C43S and G594∆ mutants were constructed by oligonucleotide-directed mutagenesis of the cDNA encoding murine APA in the vector SRαBP-1 (a gift from Professor M. D. Cooper, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, U.S.A.) [6,14] using the Stratagene QuikChange Mutagenesis Kit. The mutants generated were confirmed by DNA sequencing.…”

“…Although both APA and APN are subject to proteolytic cleavage between the two extracellular domains in i o, the resulting fragments appear to remain tightly associated with one another, and this proteolytic fragmentation does not alter the enzymic activity of the proteins [11,13]. Interestingly all the residues involved in metal binding and catalysis that have been identified through site-directed mutagenesis and\or sequence comparisons with other zinc metalloproteinases, such as thermolysin [14][15][16][17][18], are all located in the 107 kDa domain of APA. Thus it is unclear whether the 45 kDa domain is required for enzymic activity or whether it serves some other function.…”

Aminopeptidase-A. I. cDNA cloning and expression and localization in rat tissues

“…APA has been characterized as being a metalloenzyme with Zn 2ϩ in the catalytic site (29). The signature Zn-binding motif HEVLHX 18 E was shown previously to be essential for catalytic activity of human APA (28), and the amino acid sequence surrounding this region was conserved in all three species (Fig. 3).…”