Cloning, expression and characterization of low-molecular-weight acid phosphatases from bovine heart and human placenta
Abstract
Acid phosphatases (orthophosphoric-monoester phosphohydrolyases; EC 3.1.3.2) catalyze the hydrolysis of orthophosphate monoesters and the reaction has a pH optimum in the dilute acid range. Based upon the published amino acid sequence of a low-molecular-weight (18 kDa) bovine liver acid phosphatase, a full length cDNA clone coding for bovine heart low-molecular-weight acid phosphatase (BHPTP) was isolated and sequenced. By using a T7 expression system, the native bovine heart enzyme was overproduced in E. coli. The expressed BHPTP was nearly identical to the authentic enzyme in molecular weight, immunological reactivity and kinetic properties. A DNA fragment corresponding to the coding region of the isolated bovine heart cDNA clone was subsequently used as a probe to screen a human placental $\lambda$gt11 cDNA library. Several overlapping cDNA clones coding for two distinct human cytoplasmic, low molecular weight, phosphotyrosyl protein phosphatases (HCPTPs) were thus obtained. The two longest clones, HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of 108 base pair segment in the middle of the open reading frame. The gene coding for these HCPTPs was assigned to chromosome 2, which is also known to be the location of the human red cell acid phosphatase locus ACP1. Studies using genomic DNA indicate that the variable region of HCPTP1-1 and HCPTP2-1 does not result from the alternative RNA splicing. Based on RNA blotting experiments, the gene coding for both types of HCPTPs is expressed in all human tissues studied. The isoenzymes encoded by HCPTP1-1 and HCPTP2-1 were overexpressed in E. coli and the resulting recombinant enzymes (i.e. HCPTP-A and HCPTP-B) were characterized by SDS-PAGE, immunoblotting and phosphatase activity assay. The expressed proteins were strongly active towards the phosphatase substrates, p-nitrophenyl phosphate, $\beta$-naphthyl phosphate, O-phospho-L-tyrosine, phosphotyrosyl peptides and phosphotyrosyl myelin basic protein, but not $\alpha$-naphthyl phosphate, threonine phosphate, O-phospho-L-serine, or phosphoseryl and phosphothreonyl casein. HCPTP-A and HCPTP-B exhibited different substrate specificities and sensitivities towards Zn$\sp{2+}$. It is concluded that HCPTPs are in fact two members of a phosphotyrosyl protein phosphatase family, and that they may have different physiological substrates in the cell.
Degree
Ph.D.
Advisors
Etten, Purdue University.
Subject Area
Biochemistry
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