scholarly journals Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme

1990 ◽  
Vol 265 (2) ◽  
pp. 429-433 ◽  
Author(s):  
N M Hooper ◽  
J N Keen ◽  
A J Turner
Keyword(s):  
Human Kidney ◽  
Amino Acid Sequences ◽  
Kidney Cortex ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
C Terminus ◽  
The Difference ◽  
Solubilized Form ◽  
Triton X ◽  
High Degree

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.

scholarly journals Ectoenzymes of the kidney microvillar membrane. Isolation and characterization of the amphipathic form of renal dipeptidase and hydrolysis of its glycosyl-phosphatidylinositol anchor by an activity in plasma

1989 ◽  
Vol 261 (3) ◽  
pp. 811-818 ◽  
Author(s):  
N M Hooper ◽  
A J Turner
Keyword(s):  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Isolation And Characterization ◽  
C Terminus ◽  
Terminal Amino ◽  
Membrane Isolation ◽  
Solubilized Form ◽  
Triton X ◽  
Hydrolysis Of

Renal dipeptidase (EC 3.4.13.11) has been solubilized from pig kidney microvillar membranes with n-octyl-beta-D-glucopyranoside and then purified by affinity chromatography on cilastatin-Sepharose. The enzyme exists as a disulphide-linked dimer of two identical subunits of Mr 45,000 each. The purified dipeptidase partitioned into the detergent-rich phase upon phase separation in Triton X-114 and reconstituted into liposomes consistent with the presence of the glycosyl-phosphatidylinositol membrane anchor. The N-terminal amino acid sequence of the amphipathic, detergent-solubilized, form of renal dipeptidase was identical with that of the hydrophilic, phospholipase-solubilized, form, locating the membrane anchor at the C-terminus of the protein. The glycosyl-phosphatidylinositol anchor of both purified and microvillar membrane renal dipeptidase was a substrate for an activity in pig plasma which displayed properties similar to those of a previously described phospholipase D. The cross-reacting determinant of the glycosyl-phosphatidylinositol anchor was generated by incubation of purified renal dipeptidase with bacterial phosphatidylinositol-specific phospholipase c, whereas the anchor-degrading activity in plasma failed to generate this determinant.

scholarly journals cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme

1990 ◽  
Vol 271 (3) ◽  
pp. 755-760 ◽  
Author(s):  
E Rached ◽  
N M Hooper ◽  
P James ◽  
G Semenza ◽  
A J Turner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
Signal Sequence ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
Kidney Cortex ◽  
Xenopus Laevis Oocytes ◽  
Glycosyl Phosphatidylinositol ◽  
Pig Kidney ◽  
C Terminus

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.

scholarly journals Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei

1992 ◽  
Vol 283 (2) ◽  
pp. 479-485 ◽  
Author(s):  
S Pingel ◽  
M Duszenko
Keyword(s):  
Trypanosoma Brucei ◽  
Surface Glycoprotein ◽  
Buffer System ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Glycosylation Sites ◽  
Pngase F ◽  
Triton X ◽  
Variant Surface Glycoproteins

Variant surface glycoproteins (VSGs) of Trypanosoma brucei contain two distinct glycosylation sites: (1) N-linked glycans within the protein portion of the molecules, and (2) the glycosyl-phosphatidylinositol (GPI) membrane anchor. Since galactose residues show uncommon alpha-glycosidic linkages in the GPI membrane anchor, we were prompted to investigate galactosylation of the GPI anchor. On comparing a trypanosome clone galactosylated exclusively in N-glycans (clone MITat 1.5) with clones galactosylated predominantly in the glypiated membrane anchor (clones MITat 1.4, MITat 1.6 and AnTat 1.8), clone MITat 1.5 showed a 10-fold increased enzyme activity when using a protocol including Triton X-100 to assay UDPgalactose:N-acetylglucosaminyl glycopeptide beta 1,4-galactosyltransferase (EC 2.4.1.38). Only the VSG of clone MITat 1.5 could be radiochemically labelled with UDP[14C]galactose, and galactosylation of N-glycans was confirmed by digestion with peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase F). However, in a modified enzyme assay without detergent, galactosyltransferase activity was increased considerably (15-fold) in clone MITat 1.4. VSG galactosylation of clones MITat 1.4, MITat 1.6 and AnTat 1.8 was readily detected by fluorography of the respective SDS/polyacrylamide gels, suggesting that galactosyltransferase activity modifies the VSG membrane anchor in these clones. In this case, [14C]galactose labelling of immunoprecipitated VSG (clone MITat 1.4) was resistant to the release of N-glycans by PNGase F treatment, and thus revealed galactosylation in vitro of a VSG membrane anchor. Exoglycosidase digestions of VSG MITat 1.4 confirmed the presence of alpha-linked galactose residues. We suggest that these specific alpha-galactosyltransferases are inhibited by the action of detergent, but can be activated in a detergent-free buffer system.

Cloning of the Rat Tissue Factor cDNA and Promoter: Identification of a Serum-response Region

1996 ◽  
Vol 76 (05) ◽  
pp. 697-702 ◽  
Author(s):  
Olivier Taby ◽  
Claire-Lise Rosenfield ◽  
Vladimir Bogdanov ◽  
Yale Nemerson ◽  
Mark B Taubman
Keyword(s):  
Tissue Factor ◽  
General Structure ◽  
Amino Acid Sequences ◽  
Proximal Promoter ◽  
Striking Similarity ◽  
Upstream Sequence ◽  
Tf Gene ◽  
Rat Tissue ◽  
High Degree ◽  
Transcriptional Elements

SummaryTissue factor (TF) initiates coagulation and its expression in vascular smooth muscle cells (VSMC) likely plays a role in the propagation of arterial thrombosis. We report cloning the cDNA and proximal promoter region of the rat TF gene. While maintaining the general structure and organization of the TF molecule, there is a surprising divergence (≈ 18%) between the derived amino acid sequences of the rat and mouse TF. In contrast, there is striking similarity (90%) in the 5’ untranslated regions. High levels of basal promoter activity were seen in rat VSMC with constructs containing 106 bp of sequence downstream from the putative transcription start site and 426 to 103 bp of upstream sequence. Deletion of the sequence from −103 to −79, containing a single SP1 site, removed virtually all of the basal and serum-induced activity. Removal of the NFkB site or two additional upstream SP1 sites had little effect on serum responsiveness. Removal of the 5’ untranslated region abolished most of the basal activity of the TF promoter, suggesting that its high degree of conservation may be due to the presence of transcriptional elements critical for TF expression in rodent VSMC.

scholarly journals Identification of a functionally conserved surface region of rat cytochromes P450IA

1991 ◽  
Vol 278 (3) ◽  
pp. 749-757 ◽  
Author(s):  
R J Edwards ◽  
A M Singleton ◽  
B P Murray ◽  
S Murray ◽  
A R Boobis ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Cytochromes P450 ◽  
Surface Region ◽  
Carrier Protein ◽  
Peptide Antigen ◽  
Aryl Hydrocarbon ◽  
C Terminus ◽  
Aryl Hydrocarbon Hydroxylase Activity ◽  
The Difference ◽  
Peptide Antibodies

A region of rat cytochrome P450IA1 at residues 294-301 (Gln-Asp-Arg-Arg-Leu-Asp-Glu-Asn), equivalent to a proinhibitory region of cytochrome P450IA2, was identified by sequence alignment. Anti-peptide antibodies were successfully raised when the peptide was coupled through either its N- or its C-terminus to carrier protein, but no antibodies were produced against the so-called multiple peptide antigen, which consisted of eight copies of the peptide attached through its C-terminus to a synthetic base. Both of the anti-peptide antibodies bound specifically to cytochrome P450IA1 in the rat, as shown by e.l.i.s.a. and immunoblotting. They inhibited microsomal aryl hydrocarbon hydroxylase activity and the mutagenic activation of 2-acetylaminofluorene (these reactions are catalysed by cytochrome P450IA1), but not high-affinity phenacetin O-de-ethylation activity, which is catalysed by cytochrome P450IA2. However, there was differences in the properties of the two antisera in their binding to cytochromes P450IA1 in species other than the rat, their relative binding to the multiple peptide antigen, the yield of antibody following affinity purification using peptide coupled through its N-terminus to CNBr-activated Sepharose, and the binding of the purified preparations to N- and C-terminal-coupled peptide conjugates. These observations indicated that the antibodies were directed to the region of the peptide opposite to the end which was coupled to the carrier protein. Nevertheless, both of the antibody preparations bound equally well to the target cytochrome P450, thus indicating that, in the native protein, the whole of the peptide region is exposed on the surface of cytochrome P450IA1 and is available for binding by the antibodies. The role of this region appears to be the same in both cytochromes P450IA1 and P450IA2, despite the difference in its primary structure in the two cytochromes P450.

scholarly journals Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

1990 ◽  
Vol 265 (28) ◽  
pp. 16955-16964 ◽  
Author(s):  
P Schneider ◽  
M A Ferguson ◽  
M J McConville ◽  
A Mehlert ◽  
S W Homans ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol

scholarly journals Structure and expression of human IgG FcRII(CD32). Functional heterogeneity is encoded by the alternatively spliced products of multiple genes.

1989 ◽  
Vol 170 (4) ◽  
pp. 1369-1385 ◽  
Author(s):  
D G Brooks ◽  
W Q Qiu ◽  
A D Luster ◽  
J V Ravetch
Keyword(s):  
Structural Heterogeneity ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Membrane Glycoproteins ◽  
Igg Binding ◽  
Genes Expression ◽  
Alternatively Spliced ◽  
Membrane Spanning ◽  
High Degree ◽  
Membrane Spanning Domains

The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.

17-Oxidoreduction of 17β-Estradiol, Estrone and Their 3-Sulfates by Kidney Slices from Guinea Pig and Human

1975 ◽  
Vol 53 (12) ◽  
pp. 1333-1336 ◽  
Author(s):  
R. Hobkirk ◽  
Mona Nilsen ◽  
Barbara Jennings
Keyword(s):  
Guinea Pig ◽  
Marked Reduction ◽  
Human Kidney ◽  
Kidney Cortex ◽  
Limited Extent ◽  
Tissue Slices ◽  
Ample Evidence ◽  
Sulfatase Activity ◽  
17Β Estradiol ◽  
Dehydrogenation Reactions

Slices of whole kidney and kidney cortex from the female guinea pig catalyzed a marked reduction of estrone 3-sulfate (E13S) and estrone (E1) to 17β-estradiol 3-sulfate (E23S) and 17β-estradiol (E2), respectively, as well as the reverse (dehydrogenation) reactions. Slices of medulla did not appear active in E23S–E13S interconversion but did possess the ability to interconvert E2 and E1, besides possessing considerable sulfatase activity. The use of [3H-35S]E13S and [3H-35S]E23S as substrates, together with a demonstrated lack of estrogen sulfate synthesis by the tissue slices, provided ample evidence that the intact sulfates were involved in direct oxidoreduction. Slices of human kidney cortex catalyzed the reduction of E13S to a very limited extent. Slices of whole kidney and of cortex from guinea pig formed small amounts of estrogen glucuronide(s).

scholarly journals The Differential Effects of pp120 (Ceacam 1) on the Mitogenic Action of Insulin and Insulin-Like Growth Factor 1 Are Regulated by the Nonconserved Tyrosine 1316 in the Insulin Receptor

2000 ◽  
Vol 20 (11) ◽  
pp. 3896-3905 ◽  
Author(s):  
Payal Soni ◽  
Montaha Lakkis ◽  
Matthew N. Poy ◽  
Mats A. Fernström ◽  
Sonia M. Najjar
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Site Directed Mutagenesis ◽  
Insulin Like Growth Factor ◽  
Β Subunit ◽  
Conserved Domain ◽  
C Terminus ◽  
Mitogenic Action ◽  
The Difference ◽  
Differential Phosphorylation

ABSTRACT pp120 (Ceacam 1) undergoes ligand-stimulated phosphorylation by the insulin receptor, but not by the insulin-like growth factor 1 receptor (IGF-1R). This differential phosphorylation is regulated by the C terminus of the β-subunit of the insulin receptor, the least conserved domain of the two receptors. In the present studies, deletion and site-directed mutagenesis in stably transfected hepatocytes derived from insulin receptor knockout mice (IR−/−) revealed that Tyr1316, which is replaced by the nonphosphorylatable phenylalanine in IGF-1R, regulated the differential phosphorylation of pp120 by the insulin receptor. Similarly, the nonconserved Tyr1316 residue also regulated the differential effect of pp120 on IGF-1 and insulin mitogenesis, with pp120 downregulating the growth-promoting action of insulin, but not that of IGF-1. Thus, it appears that pp120 phosphorylation by the insulin receptor is required and sufficient to mediate its downregulatory effect on the mitogenic action of insulin. Furthermore, the current studies revealed that the C terminus of the β-subunit of the insulin receptor contains elements that suppress the mitogenic action of insulin. Because IR−/− hepatocytes are derived from liver, an insulin-targeted tissue, our observations have finally resolved the controversy about the role of the least-conserved domain of insulin and IGF-1Rs in mediating the difference in the mitogenic action of their ligands, with IGF-1 being more mitogenic than insulin.

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

1991 ◽  
Vol 280 (3) ◽  
pp. 745-751 ◽  
Author(s):  
N M Hooper ◽  
A Bashir
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Aqueous Phase ◽  
Hydrophobic Membrane ◽  
Rich Phase ◽  
Glycosyl Phosphatidylinositol ◽  
Ionic Detergent ◽  
Membrane Spanning ◽  
Triton X ◽  
Insoluble Pellet

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

Sign in / Sign up

Export Citation Format

Share Document