Plasma Cell Membrane Glycoprotein PC-1 Research Articles

Mechanisms of Disease: ectonucleotide pyrophosphatase phosphodiesterase 1 as a 'gatekeeper' of insulin receptors

Insulin resistance is pathogenic for type 2 diabetes and cardiovascular disease. Several inhibitors of insulin signaling have a role in human insulin resistance. The transmembrane glycoprotein ectonucleotide pyrophosphatase phosphodiesterase 1 (E-NPP1; also known as plasma cell membrane glycoprotein PC-1) interacts with the insulin receptor and inhibits subsequent signaling by decreasing its beta-subunit autophosphorylation. E-NPP1 is overexpressed in skeletal muscle, adipose tissue and cultured skin fibroblasts of insulin-resistant individuals who are not yet obese or diabetic, which indicates that excessive E-NPP1 expression is an early, intrinsic defect in human insulin resistance. Genetic studies also support a primary role of E-NPP1 in insulin resistance. Among other variants, a missense polymorphism, Lys121Gln, has been described. The Gln121 variant is a stronger inhibitor than Lys121 of insulin receptor function, and is associated with insulin resistance, type 2 diabetes and both cardiovascular and nephrovascular complications in diabetic patients. E-NPP1 is measurable in human serum, where it might represent a valuable biomarker of insulin resistance, but its relationship to tissue and systemic insulin resistance remains to be thoroughly elucidated. Understanding the mechanisms that regulate E-NPP1 expression and/or function might render this protein a new target for strategies to treat and prevent type 2 diabetes and cardiovascular disease.

Structural basis of allotypes of ecto‐nucleotide pyrophosphatase/phosphodiesterase (plasma cell membrane glycoprotein PC‐1) in the mouse and rat, and analysis of allele‐specific xenogeneic antibodies

Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) have been implicated in bone calcification, type II diabetes, control of purinergic signalling and tumour invasion. The gene for the plasma cell membrane glycoprotein PC-1 in the mouse (Enpp1) has been known since 1970 to exist in two allelic forms, but their structural basis was heretofore unknown. We show that the Enpp1a and Enpp1b alleles differ by only two amino acids, at positions 650 and 679 in the C-terminal nuclease-like domain. Histidine 650 but not arginine 679 forms an essential part of the Enpp1a epitope recognized by monoclonal antibody IR-518. Sequences of LEW and LOU rats and the rat glioma cell line C6 differ from that of the mouse by about 60 amino acids. The LOU and C6 cell line sequences differ by only three amino acids, but differ from the LEW sequence by 10 amino acids. All three rat strains possess the mouse Enpp1b allele at positions 650 and 679. Despite numerous other differences from the mouse, rats immunized with Enpp1a mouse cells have generated monoclonal antibodies specific for the Enpp1a allele, suggesting that amino acids 650 and 679 may be particularly immunogenic. The cytoplasmic tails of the mouse and rat are highly conserved, but are significantly different from human cytoplasmic tails.

A Major Locus for Fasting Insulin Concentrations and Insulin Resistance on Chromosome 6q with Strong Pleiotropic Effects on Obesity-Related Phenotypes in Nondiabetic Mexican Americans

Insulin resistance and hyperinsulinemia are strong correlates of obesity and type 2 diabetes, but little is known about their genetic determinants. Using data on nondiabetics from Mexican American families and a multipoint linkage approach, we scanned the genome and identified a major locus near marker D6S403 for fasting "true" insulin levels (LOD score 4.1, empirical P<.0001), which do not crossreact with insulin precursors. Insulin resistance, as assessed by the homeostasis model using fasting glucose and specific insulin (FSI) values, was also strongly linked (LOD score 3.5, empirical P<.0001) with this region. Two other regions across the genome were found to be suggestively linked to FSI: a location on chromosome 2q, near marker D2S141, and another location on chromosome 6q, near marker D6S264. Since several insulin-resistance syndrome (IRS)-related phenotypes were mapped independently to the regions on chromosome 6q, we conducted bivariate multipoint linkage analyses to map the correlated IRS phenotypes. These analyses implicated the same chromosomal region near marker D6S403 (6q22-q23) as harboring a major gene with strong pleiotropic effects on obesity and on lipid measures, including leptin concentrations (e.g., LOD(eq) for traits-specific insulin and leptin was 4.7). A positional candidate gene for insulin resistance in this chromosomal region is the plasma cell-membrane glycoprotein PC-1 (6q22-q23). The genetic location on chromosome 6q, near marker D6S264 (6q25.2-q26), was also identified by the bivariate analysis as exerting significant pleiotropic influences on IRS-related phenotypes (e.g., LOD(eq) for traits-specific insulin and leptin was 4.1). This chromosomal region harbors positional candidate genes, such as the insulin-like growth factor 2 receptor (IGF2R, 6q26) and acetyl-CoA acetyltransferase 2 (ACAT2, 6q25.3-q26). In sum, we found substantial evidence for susceptibility loci on chromosome 6q that influence insulin concentrations and other IRS-related phenotypes in Mexican Americans.

Divalent cations stabilize the conformation of plasma cell membrane glycoprotein PC-1 (alkaline phosphodiesterase I).

The plasma cell-membrane glycoprotein PC-1 is an ectoenzyme with alkaline phosphodiesterase I/5'-nucleotide phosphodiesterase (EC 3.1.4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It contains sequence motifs which closely match the consensus EF-hand (helix-loop-helix) Ca(2+)-binding regions of parvalbumin, troponin-C and calmodulin, and its enzymic activity is increased in the presence of divalent cations and decreased in the presence of chelating agents. We have undertaken experiments to determine whether divalent cations affect the conformation of the PC-1 protein, as assessed by their effect on thermal stability, resistance to proteolysis and binding of polyclonal antibodies to the whole native protein and monoclonal antibodies to a putative Ca(2+)-binding region. Divalent cations were found to protect solubilized PC-1 against thermal denaturation and proteolysis. They also stabilized PC-1 on intact cells; this form was much more resistant to proteolysis than Triton X-100 solubilized PC-1. Ca2+, Mg2+ and Zn2+ ions were equally effective. Monoclonal antibodies to the bacterially expressed C-terminal EF-hand homology region only bound to mammalian PC-1 in the absence of Ca2+. In contrast, the great majority of polyclonal antibodies to native PC-1 bound regardless of whether Ca2+ was present or not, but with increased binding when Ca2+ was present. These results provide evidence that divalent cations bind to PC-1 and stabilize its conformation.

Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells. Regulation of PC-1 expression in osteosarcoma cells by transforming growth factor-beta.

A bone and cartilage enzyme with both 5'-nucleotide phosphodiesterase I and nucleotide pyrophosphohydrolase (NTPPPH) activity modulates physiologic mineralization and pathologic chondrocalcinosis by generating inorganic pyrophosphate. We hypothesized that, as for alkaline phosphatase, expression of an NTPPPH gene can be shared by cells from bone, cartilage, and liver and by certain leukocytes. Recently, we demonstrated the hepatocyte and murine plasma cell membrane glycoprotein PC-1 to have both 5'-nucleotide phosphodiesterase I and NTPPPH activity. We detected polypeptides cross-reactive with PC-1 in human U20S osteosarcoma cells, articular chondrocytes, homogenized human knee cartilages, human knee synovial fluids, hepatoma cells, and murine plasmacytoma cells. Constitutive low abundance PC-1 mRNA expression was detected in U20S cells and chondrocytes by a nested RNA-PCR assay and by Northern blotting. TGF beta is known to substantially increase NTPPPH activity in primary osteoblast cultures. We demonstrated that TGF beta 1 increased NTPPPH activity and the level of PC-1 mRNA and immunoprecipitable [35S]-methionine-labeled PC-1 polypeptides in U20S cells. The identification of PC-1 as an NTPPPH expressed in cells derived from bone and cartilage may prove useful in furthering the understanding of the role of NTPPPH i n physiologic and pathologic mineralization.

Molecular cloning of cDNAs for human fibroblast nucleotide pyrophosphatase

Nucleotide pyrophosphatase (NPPase) activity is markedly elevated in cultured skin fibroblasts from patients of Lowe's syndrome. cDNA clones encoding the NPPase were isolated using synthetic oligonucleotide probes designed on the basis of partial amino acid sequence of the enzyme purified from human placenta. The complete sequences of these clones yielded a merged sequence of 3508 bases. The polypeptide chain of the enzyme was deduced to comprise 873 amino acids with a calculated molecular weight of 99,703 and had the characteristics of a class II transmembrane protein. Ten potential N-glycosylation sites were detected in the protein. RNA blot analysis showed that human fibroblasts contain two minor mRNAs of 7.0 and 8.2 kb, respectively, in addition to a major 3.6-kb species that coincides with the merged cDNA in size. A computer search of a nucleotide sequence database revealed that plasma cell membrane glycoprotein PC-1, whose function was unknown at the time, is identical with the NPPase. Comparison of the amino acid sequence of the NPPase with the active site sequence of bovine 5′-nucleotide phosphodiesterase allowed the assignment of a putative active site domain to the central region of the COOH-terminal extracellular domain of the NPPase. The gene for human NPPase was localized to chromosome 6 at q22–q23 by in situ hybridization with a fragment of the NPPase cDNA.

Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location.

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.

Detecting distant homologies of mosaic proteins: Analysis of the sequences of thrombomodulin, thrombospondin complement components C9, C8α and C8β, vitronectin and plasma cell membrane glycoprotein PC-1

Recognition of homologies may give hints about the structure and function of proteins; therefore, we are developing strategies to aid sequence comparisons. Detecting homology of mosaic proteins is especially difficult since the modules constituting these proteins are usually distantly related and their homology is not readily recognized by conventional computer programs. In the present work we show that the rules of the evolution of mosaic proteins can guide the identification of modules of mosaic proteins and can delineate the group of sequences in which the presence of homologous sequences may be expected. By this approach we can concentrate the search for homology to a limited group of sequences; thus ensuring a more intense and more fruitful search. The power of this approach is illustrated by the fact that it could detect homologies not identified by earlier methods of sequence comparison. In this paper we show that thrombomodulin contains a domain homologous with animal lectins, that complement components C9, C8α and C8β have modules homologous with one of the repeat units of thrombospondin and that the somatomedin B module of vitronectin is homologous with the internal repeats of plasma cell membrane glycoprotein PC-1.

Plasma cell membrane glycoprotein PC-1. Primary structure deduced from cDNA clones.

The PC-1 protein is a membrane glycoprotein that is selectively expressed on the surface of antibody-secreting cells. Previous work has shown that it consists of two apparently identical disulfide-bonded polypeptides, each of molecular weight approximately 120,000. We now describe the sequence of PC-1 mRNA and protein. The PC-1 protein is shown to consist of 905 amino acids and to have an uncommon transmembrane orientation. The NH2-terminal 58 residues are intracellular and the COOH-terminal 826 residues are extracellular. A cysteine-rich region of 85 amino acids lies adjacent to the extracellular surface of the membrane and appears to have arisen by exon duplication. In common with other membrane glycoproteins with this orientation, there is no obvious signal sequence other than the transmembrane segment. The PC-1 protein therefore has an overall structure and membrane orientation that resembles those of the transferrin receptor, the asialoglycoprotein receptor, and the Ia invariant chain.