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Query: UNIPROT:P06889 (Mol)
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In unstimulated mammary epithelial cells from virgin mice, the prolactin receptor exists as two isoforms: a 78 and a 70 kDa species. Both proteins are reduced to a single 61 kDa molecule after N-glycanase F treatment, indicating that their size difference is solely a result of carbohydrate content. Membrane fractionation experiments reveal that the smaller species is exclusively intracellular, while the larger one is located on the cell surface. Nitric oxide (NO) stimulates the migration of prolactin receptors from an internal pool to the plasmalemma in only 30 min and this redistribution is associated with an increase in molecular weight. Redistribution is blocked by swainsonine, but not by castanospermine or 1-deoxymannojirimycin, suggesting that the glycosylation step involved with translocation is either alpha-mannosidase II or N-acetylglucosamine (NAG) transferase I. The former is unaffected by NO but the activity of the latter is doubled 30 min after exposure to NO. These data suggest that prolactin receptors are retained intracellularly because of their incomplete N-glycosylation and that NO triggers their redistribution by stimulating the completion of this process, in part by increasing NAG transferase I activity.
Mol Cell Endocrinol 1999 Mar 25
PMID:Regulation of prolactin receptor glycosylation and its role in receptor location. 1037 21

Human FcepsilonRII/CD23 is an approximately 45 kDa type II transmembrane glycoprotein belonging to the C-type animal-lectin family, and has two isoforms (a and b) that only differ in their intracytoplasmic tails. We previously found that in several human and mouse cell lines there were two additional CD23 transcripts (a' and b') lacking the exon 3 that encodes the entire transmembrane segment and a part of cytoplasmic tails. In this study, we analyzed the putative CD23a' and CD23b' products at protein levels and characterized with rabbit polyclonal antibodies against novel amino-acid sequences of the putative CD23a' and CD23b' molecules (anti-CD23a' Ab, anti-CD23b' Ab). Western blots in COS cells transfected with CD23a' or CD23b' cDNA as well as in vitro translation assays showed that the a' and b' CD23 transcripts were translated to about 40 kDa molecules. These 40 kDa molecules were also recognized by a polyclonal antibody against 25 kDa soluble fragment of human CD23. We also found that human cells having mRNAs for CD23a' and CD23b' expressed protein products recognized specifically by anti-CD23a' or anti-CD23b' Ab, respectively. In addition, the CD23a' and CD23b' molecules in transfected COS cells were resistant to Endo H(f) and PNGase F, although these truncated forms as well as the membrane-associated forms had an asparagine residue responsible for the N-linked glycosylation. Taken together, our results show that the a' and b' CD23 transcripts are expressed and translated in human lymphoid cells and that their translated products are retained in the cytoplasm where they might play an unique regulatory role in the expression of the full-length CD23 on the cell surface.
Mol Immunol 1999 Dec
PMID:Characterization of novel FcepsilonRII/CD23 isoforms lacking the transmembrane (TM) segment in human cell lines. 1068 62

Variant splicing of gonadotropin receptor mRNA commonly occurs, however expression of receptor protein variants and their trafficking has yet to be studied in detail. To determine receptor variant trafficking and intracellular processing in mammalian cells, the intracellular fate of intentionally truncated variants of human follicle stimulating hormone receptor (hFSH-R) expressed in CHO cells was examined. Monoclonal antibodies (mAbs) were made against the hFSH-R's extracellular domain (ECD) expressed in insect cells. Four mAbs 106.156, 106.290, 106.318, and 106.263 were chosen as probes. Epitope mapping using synthetic peptides, and truncated hFSH-R variants revealed that mAb 106.156 bound to ECD residues 183-220, while mAbs 106.318, 106.290, 106.263 bound ECD residues 300-331. Immunofluorescence microscopy showed that mAbs 106.318 and 106.156 stained the surface of fixed, intact CHO cells expressing wild type hFSH-R. However, following cell permeabilization all four antibodies stained hFSH-R in Golgi and endoplasmic reticulum. Permeabilized cells expressing truncated variants ECD213 and ECD254 showed staining accumulated in the endoplasmic reticulum/nuclear envelope continuum. ECD335/His was found to accumulate in extended endoplasmic reticulum (ER). The ER location of ECD335/His was confirmed by double labeling experiments with concanavalin A and ECD mAb. Glycosidase digestion followed by Western blot analysis show ECD213 and ECD335/His to be glycosylated, but not ECD254. Both glycosylated truncated hFSH-R variants were sensitive to peptide-N-glycanase F and endoglycosidase H but insensitive to neuraminidase indicating that these variants possess high mannose type oligosaccharides. Thus truncated hFSH-R variants do not reach the medial or trans Golgi where high mannose oligosaccharides are trimmed and sialic acid is added. These data suggest that the conformation the ECD of the wild type receptor is different from the ECD alone expressed in the endoplasmic reticulum. This information suggests that the ECD serves two distinct roles; the first is to bind FSH and the other is likely to contact the endodomain of the receptor, which presumably leads to activation of the endodomain for signal transduction.
Mol Cell Endocrinol 2000 Feb 25
PMID:Human follicle stimulating hormone receptor variants lacking transmembrane domains display altered post-translational conformations. 1071 54

D-Glucose entry into erythrocytes from adult grey-headed flying fox fruit bats (Pteropus poliocephalus) was rapid and showed saturation at high substrate concentrations. Kinetic parameters were estimated from the concentration dependence of initial rates of zero-trans D-glucose entry at 5.5 degrees C as Michaelis constant (K(m)) 1. 64+/-0.56 mM, and maximal velocity (V(max)) 1162+/-152 micromol.l. cell water(-1).min(-1). D-Glucose entry was inhibited by cytochalasin B; mass law analysis of D-glucose-displaceable cytochalasin B binding gave values of K(d) 37.1+/-5.0 nM and B(max) 361.2+/-9.1 pmol/mg membrane protein. Entry of 2-deoxy-D-glucose, and 3-O-methyl-D-glucose, into P. poliocephalus red cells was rapid, entry of D-fructose was very slow. Glucose transporter polypeptides were identified on immunoblots as a band M(r) 47000-54000 and their identity confirmed by D-glucose-sensitive photolabeling of membranes with [3H]-cytochalasin B. Peptide-N-glycanase F digestion of both human and bat erythrocyte membranes generated GLUT-1-derived bands M(r) 37000. Trypsin digestion of human and fruit bat erythrocyte membranes generated fragmentation patterns consistent with similar GLUT-1 polypeptide structures in both species. Erythrocytes from adult Australian ghost bats (Macroderma gigas), a carnivorous microchiropteran bat, also expressed high levels of GLUT-1.
Comp Biochem Physiol A Mol Integr Physiol 2000 May
PMID:Rapid GLUT-1 mediated glucose transport in erythrocytes from the grey-headed fruit bat (Pteropus poliocephalus). 1090 51

Saccharomyces cerevisiae possesses three related ammonium transporters, Mep1, Mep2 and Mep3, differing in their kinetic properties and in the level and regulation of their gene expression. The three Mep proteins belong to a family conserved in bacteria, plants and animals, which also includes proteins of the rhesus blood group family. In addition to its role in scavenging extracellular ammonium, the Mep2 protein has been proposed to act as an ammonium sensor, essential to pseudohyphal differentiation in response to ammonium limitation. To pursue the biochemical study of the Mep transporters, we raised polyclonal antibodies against the C-terminal tail of each Mep protein. When electrophoresed on SDS-polyacrylamide gel, the Mep1 and Mep3 proteins migrate as expected from their predicted size, whereas the Mep2 protein migrates as a high-molecular-weight smear. Protein deglycosylation with peptide-N-glycosidase F (PNGase F) indicates that, in contrast to Mep1 and Mep3, Mep2 is an asparagine-linked glycoprotein. Site-directed mutagenesis of the four potential N-glycosylation sites of Mep2 shows that Asn-4 of the protein's N-terminal tail is the only site that binds oligosaccharides. This provides evidence for the extracytosolic location of the Mep2 N-terminus. Consistently, treatment of intact protoplasts with proteinase K leads to specific proteolysis of the N-terminal tail of Mep2. The protein's C-terminus, on the other hand, is protected against protease degradation under these conditions, but digested after protoplast permeabilization, suggesting a cytoplasmic location for this part of the protein. Mep2 glycosylation is not required for pseudohyphal differentiation in response to ammonium starvation, and its absence causes only a slight reduction in the affinity of the transporter for its substrate.
Mol Microbiol 2000 Nov
PMID:In vivo N-glycosylation of the mep2 high-affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N-terminus. 1106 79

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.
Insect Biochem Mol Biol 2001 Feb
PMID:Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands. 1116 37

Human seminal plasma alpha-L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83,000 nmol/min/mg protein) by affinity chromatography on agarose-epsilon-aminocaproyl-fucopyranosylamine. The purified alpha-L-fucosidase appeared to contain a single subunit of 56-57 kDa (as determined by SDS-PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma alpha-L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-alpha-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0-4.5) with a second neutral optimum (pH 6.4-7.4) with 60-80% of maximal activity. Apparent K(M) and V(max) values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 micromol/min/mg protein respectively, using Lineweaver-Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma alpha-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5-7. The acidic isoforms between pI values 5-6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.
Mol Hum Reprod 2002 Mar
PMID:Purification and characterization of human seminal plasma alpha-L-fucosidase. 1187 Feb 29

The Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCL) belongs to the neuronal ceroid lipofuscinosis group of common recessively inherited neurodegenerative disorders. The CLN 5 gene responsible for this brain disorder codes for a novel protein with no homology to previously reported proteins. In this study, we have investigated the biosynthesis and intracellular localization of this protein in transiently transfected BHK-21 cells using a CLN5-specific peptide antibody. Confocal immunofluorescence microscopy showed that wild-type CLN5 is predominantly targeted to lysosomes and immunoprecipitation analysis recognized a 60 kDa polypeptide. The molecular weight of this protein was reduced to 40 kDa by deglycosylation with Endo H and to 38 kDa with PNGase F. The same-sized glycosylated polypeptides were also observed in the media, suggesting that the 60 kDa glycosylated CLN5 polypeptide represents a soluble lysosomal glycoprotein, not an integral transmembrane protein as predicted earlier. The most common human vLINCL mutation blocked the lysosomal targeting of expressed polypeptides. This would imply that the pathogenesis of vLINCL would be associated with the defective lysosomal trafficking, preventing the normal biological function of the corresponding polypeptide.
Hum Mol Genet 2002 Apr 15
PMID:Lysosomal localization of the neuronal ceroid lipofuscinosis CLN5 protein. 1197 70

We have cloned and characterized a novel glucose transporter (GLUT11) that is alternatively spliced. The GLUT11 gene maps to chromosome 22q11.2 and consists of 13 exons. The long form (GLUT11-L) cDNA uses 13 exons to produce a protein containing 503 amino acids. The short form of GLUT11 (GLUT-11) cDNA is missing exon 2 and produces a protein of 496 amino acids with a 14 amino acid N-terminal difference compared to the long form. GLUT11 has significant similarity to known GLUTs and contains 12 putative membrane-spanning helices along with sugar transporter signature motifs that have previously been shown to be essential for transport activity. The putative glycosylation site of GLUT11 is present in loop 1. Northern blot analysis showed that GLUT11 mRNA is expressed in a number of tissues and most abundantly in the skeletal muscle and heart. RT-PCR assay showed that GLUT11 is alternatively spliced and the two isoforms are distributed differently in various tissues. Immunofluorescence microscopy demonstrated that GLUT11-L resides on the plasma membrane when overexpressed in HEK293T cells. Western blot analysis revealed that GLUT11-L runs as a broad band of approximately 42 kDa that was converted to a 38 kDa polypeptide by PNGase F digestion. Furthermore, a liposome reconstitution functional assay showed that GLUT11-L has glucose transport activity.
Mol Genet Metab 2002 May
PMID:Cloning and characterization of glucose transporter 11, a novel sugar transporter that is alternatively spliced in various tissues. 1217 79

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.
Mol Cell Proteomics 2002 Jul
PMID:High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies. 1223 77


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