Biochemical Characterization of Human PC‐1, an Enzyme Possessing Alkaline Phosphodiesterase I and Nucleotide Pyrophosphatase Activities

Abstract

) -EJB 94 0666/4 PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities. In this paper, we demonstrate the expression, biochemical characterization and biosynthesis of human PC-1. Previously, there has been uncertainty concerning which of two methionine residues is the initiator. It is now shown that expression of PC-1 is much greater if the first methionine residue is present, and that the sequence between the two methionine residues is translated in both human and mouse, in both transfected cells and cells naturally expressing PC-1. The first methionine residue is therefore the initiator. Human PC-1 is capable of autophosphorylation, and conditions are described in which PC-1 is the only labelled phosphoprotein on the plasma membranes of intact cells, allowing the demonstration that the mature membrane form of human PC-1 is approximately 10 kDa larger than that of the mouse form. Pulse-chase biosynthetic studies and treatment with two different endoglycosidases show that most of this difference is due to N-linked oligosaccharides. The polypeptide backbone of human PC-1 is 20 amino acids longer than that of the mouse PC-1, with most of the difference in polypeptide chain length being in the cytoplasmic domain. The revised cytoplasmic domain of human PC-1 has 76 amino acids, while the mouse cytoplasmic domain has 58 amino acids. Optimal alignment of mouse and human cytoplasmic domains reveals areas of sequence conservation in which the third bases vary. It is suggested that these regions of conservation may point to functionally important sequences in the cytoplasmic domain.PC-1 is a disulphide-bonded homodimeric type-I1 transmembrane glycoprotein which has recently been shown to be identical to both alkaline phosphodiesterase I and nucleotide pyrophosphatase [1, 21. It is expressed on plasma cells, the distal convoluted tubule of the kidney, epididymis, salivary gland ducts, capillary endothelium in the brain, and chondro-

Cellular function and molecular structure of ecto-nucleotidases

“…They share 40-50 % identity at the protein level. NPP1 was first identified as a surface-located plasma cell differentiation antigen (PC-1) of mouse B lymphocytes [392] and subsequently further characterized [393] and cloned and sequenced from mouse [394,395] and human cDNA libraries [396,397]. NPP3 was identified as the target of the monoclonal antibody RB13-6 in rat brain glial precursor cells and cloned from rat [398] and a human prostate cDNA library [399].…”

Ecto-enzymes: physiology meets pathology

“…The lengths of the tails are different (58 and 76 amino acids, respectively), and it is necessary to introduce three large gaps to maximize the homology. This discrepancy led to some confusion concerning the identity of the initiator methionine, but it is now definitively established that the first methionine in each sequence is the initiator [92]. In spite of the differences in length of the cytoplasmic tails, there are ''islands'' of very strong conservation, presumably reflecting functional constraints.…”

Differential effects of aging on human chondrocyte responses to transforming growth factor β. Increased pyrophosphate production and decreased cell proliferation

“…PPi generation is a byproduct of many synthetic reactions in the cell (9) and is a direct product of enzymes that have nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity (EC 3.6.1.8). These enzymes are concentrated in fractions enriched in plasma membranes (lo), and cleave the phosphodiester 1 bond of purine and pyrimidine nucleoside triphosphates (2, 11,12). PPi degradation is affected by several inorganic pyrophosphatases, including alkaline phosphatase (9,13).…”