Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.     

Mutational analysis of the role of N-glycosylation in alpha-factor receptor function

The alpha-factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells. The alpha-factor receptor is known to undergo several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation. Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha-factor receptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites. Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated. The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity. However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures. These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases.     

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.     

Characterization of membrane association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein

Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.     

Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix

The NTB-VPg protein of Tomato ringspot nepovirus is an integral membrane protein found in association with endoplasmic reticulum (ER)-derived membranes active in virus replication. A transmembrane helix present in a hydrophobic region at the C terminus of the NTB domain was previously shown to traverse the membranes, resulting in the translocation of the VPg domain in the lumen. We have now conducted an in planta analysis of membrane-targeting domains within NTB-VPg using in-frame fusions to the green fluorescent protein (GFP). As expected, the entire NTB-VPg protein directed the GFP fluorescence to ER membranes. GFP fusion proteins containing the C-terminal 86 amino acids of NTB-VPg also associated with ER membranes, resulting in ER-specific glycosylation at a naturally occurring glycosylation site in the VPg domain. Deletion of the hydrophobic region prevented the membrane association. The N-terminal 80 amino acids of NTB were also sufficient to direct the GFP fluorescence to intracellular membranes. A putative amphipathic helix in this region was necessary and sufficient to promote membrane association of the fusion proteins. Using in vitro membrane association assays and glycosylation site mapping, we show that the N terminus of NTB can be translocated in the lumen at least in vitro. This translocation was dependent on the presence of the putative amphipathic helix, suggesting that oligomeric forms of this helix traverse the membrane. Taken together, our results suggest that at least two distinct elements play a key role in the insertion of NTB-VPg in the membranes: a C-terminal transmembrane helix and an N-terminal amphipathic helix. An updated model of the topology of the protein in the membrane is presented.     

Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation.

Increased expression of P-glycoprotein (Pgp) has been demonstrated to cause multidrug resistance (MDR) in vitro, and it may be responsible for chemotherapy failure in a number of human cancers. Pgp is a plasma membrane protein thought to function as an energy-dependent drug transporter. From its deduced protein sequence the topology of Pgp was proposed to contain 12 transmembrane domains with six extracellular loops and two cytoplasmic ATP-binding sites. To investigate further the membrane orientation of Pgp, we have expressed a full length cDNA of mouse mdr1, as well as its truncated forms, in a cell-free system supplemented with dog pancreatic microsomal membranes (RM). We determined which domains of the in vitro-synthesized Pgp had transversed the RM membranes by analyzing their resistance to protease digestion and their glycosylation state. To our surprise, this system revealed that a significant portion of in vitro-synthesized Pgp molecules has an additional glycosylated domain in the C-terminal half. Previously, only the first predicted extracellular loop near the N terminus had been thought to be glycosylated. Furthermore, we discovered that Pgp has at least two functional signal recognition particle/docking protein dependent signal sequences, one at the N-terminal half and the other at the C-terminal half. These findings suggest a new topological model for in vitro synthesized P-glycoprotein which may be relevant to its in vivo topology.     

ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space

Mutations in the ATP-binding cassette transporter A1 (ABCA1) transporter are associated with Tangier disease and a defect in cellular cholesterol efflux. The amino terminus of the ABCA1 transporter has two putative in-frame translation initiation sites, 60 amino acids apart. A cluster of hydrophobic amino acids form a potentially cleavable signal sequence in this 60-residue extension. We investigated the functional role of this extension and found that it was required for stable protein expression of transporter constructs containing any downstream transmembrane domains. The extension directed transporter translocation across the ER membrane with an orientation that resulted in glycosylation of amino acids immediately distal to the signal sequence. Neither the native signal sequence nor a green fluorescent protein tag, fused at the amino terminus, was cleaved from ABCA1. The green fluorescent protein fusion protein had efflux activity comparable with wild type ABCA1 and demonstrated a predominantly plasma membrane distribution in transfected cells. These data establish a requirement for the upstream 60 amino acids of ABCA1. This region contains an uncleaved signal anchor sequence that positions the amino terminus in a type II orientation leading to the extracellular presentation of an approximately 600-amino acid loop in which loss-of-function mutations cluster in Tangier disease patients.     

The effect of cryogenic storage on human erythrocyte membrane proteins as determined by polyacrylamide-gel electrophoresis

A study was conducted to determine the effects of freezing on the major membrane proteins of isolated human erythrocyte membranes. Membranes in low or normal ionic strength medium were frozen at slow or fast freezing rates. The membrane protein composition and elution of proteins from the membranes were studied utilizing polyacrylamide-gel electrophoresis in a sodium dodecyl sulfate or an acetic acid-urea-phenol solvent system. Neither a change in the composition of the membrane proteins nor any elution of membrane protein during freezing and thawing was observed. The data indicate that any human erythrocyte membrane damage during freezing and thawing was not related to a change in major membrane protein composition. Human red cell membranes were stable at ?80 or ?196 °C in the absence of a cryoprotective agent.     

Membrane Association and Multimerization of Secreton Component PulC

The PulC component of the Klebsiella oxytoca pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.     

High affinity binding of an N-terminal myristoylated p60src peptide

N-Myristoyl and non-myristoyl peptides corresponding to the N terminus of p60src were used to examine whether N-myristoylation facilitates the binding of p60src to specific protein sites at the plasma membrane. We discovered high affinity protein acceptor sites (Kd = 2.7 nM) to a 15-amino acid N-myristoylated N-terminal p60src peptide in red cell membrane vesicles. Binding was not competed by the non-myristoylated analog of the peptide nor by shorter N-myristoyl src peptides and peptides homologous to the N terminus of other N-myristoylated proteins. Binding was not evident after treatment of vesicles with proteolytic enzymes. Raising the salt concentration of the buffer to 50 mM NaCl caused an apparent inhibition of binding. However, no significant effect of salt was observed on the off-rate of bound ligand under these conditions. The results indicate the existence of N-myristoyl-dependent p60src protein acceptor sites at or near the plasma membrane/skeleton interface of red cells which could be responsible for the localization of p60src to this region and may represent new regulatory components for p60src-mediated tyrosine kinase activity.     

Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.     

Secretion and membrane integration of a filamentous phage-encoded morphogenetic protein

The filamentous phage-encoded gene IV protein is required at high levels for virus assembly, although it is not a constituent of the virion. It is an integral membrane protein that does not contain an extended hydrophobic region of the kind often required for stable integration in the inner membrane. Rather, like a number of Escherichia coli outer membrane proteins, pIV is rich in charged amino acid residues and is predicted to consist of extensive beta-sheet structures. In phage-producing cells, pIV is primarily detected in the outer membrane, while in cells that produce it from the cloned gene, pIV is found in both the inner and outer membranes. The protein is synthesized as a precursor. Following cleavage of the signal sequence and translocation into the periplasm, the mature form is initially found as a soluble species. Soluble pIV then integrates into the membrane with a half-time of one to two minutes. Neither phage assembly nor other phage proteins are needed for this membrane integration, and phage assembly does not require the presence of the soluble form. The gene IV protein may be part of the structure through which the assembling phage is extruded.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.     

大熊猫犬瘟热病毒附着或血凝蛋白基因的序列分析

首次对犬瘟热病毒（ＣＤＶ）大熊猫（ＧＰ）毒株附着或血凝蛋白（Ｈ）基因进行了序列测定并与疫苗株Ｏｎｄｅｒｓｔｅｐｏｏｒｔ进行了比较。我们设计合成了４对引物,对ＧＰ株进行了ＲＴ－ＰＣＲ扩增与测序。Ｈ蛋白基因全长为１９４６ｂｐ,开放阅读框架（ＯＲＦ）始于２１－２３位的ＡＴＧ,终止于１８４２－１８４４位的ＴＧＡ,编码６０７个氨基酸,该基因序列已被ＧｅｎＢａｎｋ。将ＧＰ毒株与ＧｅｎＢａｎｋ中疫苗弱毒株Ｏｎｄ     

N glycosylation of the virus binding domain is not essential for function of the human poliovirus receptor.

The human poliovirus receptor (hPVR) is a glycoprotein with three immunoglobulin-like extracellular domains, of which the N-terminal domain (V-type domain) is necessary and sufficient for virus binding and uptake. The effect of N glycosylation of the V domain of hPVR on binding and entry of poliovirus was studied. Stable mouse L-cell lines were generated that express PVR-specific cDNA. One of the cell lines expressed a mutant of hPVR, in which both asparagine residues of the two N-glycosylation sites of the V domain were changed to aspartate (N105D) and serine (N120S), respectively. In the second mutant cell line, the portion of the cDNA encoding the V domain of hPVR was substituted by the homologous sequence of the recently isolated PVR cDNA from monkey cells. This V domain naturally lacks both N glycosylation sites and encodes D105 and S120 at the respective positions of the open reading frame. Absence of N glycosylation at these sites was demonstrated by in vitro translation of the two mutant coding sequences in the presence of microsomal membranes. Both PVR mutant cell lines were capable of poliovirus binding and replication. However, binding of anti-PVR monoclonal antibody D171 and protection from viral replication by this antibody were observed only with the glycosylation mutant carrying the human V domain. In contrast, infection of the cell line expressing the monkey-human hybrid receptor was not blocked even though monkey cells are fully protected by monoclonal antibody D171. The data suggest that N glycosylation of the V domain of hPVR is not essential for viral replication in human tissues and that differential glycosylation of hPVR at these sites is likely not a determinant of viral tissue tropism. Furthermore, the virus binding site and the epitope recognized by monoclonal antibody D171 do not appear to overlap.     

Nascent chicken ovalbumin contains the functional equivalent of a signal sequence

Highly purified mRNA for chicken ovalbumin has been translated in a cell-free protein synthesizing system from rabbit reticulocytes in the presence or absence of EDTA-stripped microsomal membranes from dog pancreas. Nascent--but not completed--ovalbumin was transferred across the microsomal membrane, as demonstrated by cotranslational core glycosylation of ovalbumin nascent chains, by resistance to posttranslational proteolysis of only the glycosylated ovalbumin chains, and by cosedimentation with the membrane of exclusively the glycosylated form. Furthermore, nascent chains of bovine prolactin were observed to compete with nascent ovalbumin for transfer across the microsomal membrane. However, no competition for membrane sites was observed between nascent chains of rabbit globin and either nascent ovalbumin or prolactin. We interpret these results to suggest that nascent ovalbumin contains the functional equivalent of a signal sequence for transfer across membranes, and that membrane components involved in the segregation of secretory proteins with cleaved signal sequences also function in the segregation of ovalbumin.     

Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation

Wild-type (WT) Yarrowia lipolytica strain secretes a major extracellular lipase Lip2p which is glycosylated. In silico sequence analysis reveals the presence of two potential N-glycosylation sites (N113IS and N134NT). Strains expressing glycosylation mutant forms were constructed. Esterase activities for the different forms were measured with three substrates: p-nitrophenol butyrate (p-NPB), tributyrin and triolein. Sodium dodecyl sulfate polacrylamide gel electrophoresis analysis of supernatant indicated that the suppression of the two sites of N-glycosylation did not affect secretion. S115V or N134Q mutations led to lipase with similar specific activity compared with WT lipase while a T136V mutation reduced specific activity toward p-NPB and tributyrin. Electrospray ionization MS of the WT entire protein led to an average mass of 36 950 Da, higher than the mass deduced from the amino acid sequence (33 385 Da) and to the observation of at least two different mannose structures: Man(8)GlcNAc(2) and Man(9)GlcNAc(2). LC-tandem MS analysis of the WT Lip2p after trypsin and endoproteinase Asp-N treatments led to high coverage (87%) of protein sequence but the peptides containing N113 and N134 were not identified. We confirmed that the presence of N-glycosylation occurred at both N113 and N134 by MS of digested proteins obtained after enzymatic deglycosylation or from mutant forms.