The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency.

Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein.

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N-glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.

Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors.

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa=Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa=Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency.

N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.

The hydroxy amino acid in an Asn-X-Ser/Thr sequon can influence N-linked core glycosylation efficiency and the level of expression of a cell surface glycoprotein.

N-Linked glycosylation usually occurs at the sequon, Asn-X-Ser/Thr. In this sequon, the side chain of the hydroxy amino acid (Ser or Thr) may play a direct catalytic role in the enzymatic transfer of core oligosaccharides to the Asn residue. Using recombinant variants of rabies virus glycoprotein (RGP), we examined the influence of the hydroxy amino acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser sequon at Asn37 was modified by site-directed mutagenesis to change the sequon to either Asn-X-Cys or Asn-X-Thr. The impact of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glycosylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Thr dramatically increases core glycosylation efficiency of Asn37 in both membrane-anchored and secreted forms of RGP. The substitution of Thr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the sequon at Asn37 is the only sequon in the protein. Novel forms of membrane-anchored and secreted RGP which are fully glycosylated at all three sequons were also generated by substitution of Thr at position 39.

DNA typing of the human MN and Ss blood group antigens in amniotic fluid and following massive transfusion.

Although red blood cell (RBC) antigen typing by agglutination is generally useful, several situations exist where this approach is difficult or impossible. For example, following a massive transfusion, a patient's residual RBCs are mixed with transfused normal donor RBCs. In this case, typing by hemagglutination primarily detects the antigens on the heterogeneous population of transfused RBCs. Agglutination testing is also of limited use for determining the phenotype of a fetus at risk for hemolytic disease of the newborn because fetal RBCs must be obtained by periumbilical blood sampling. Determining the genotype of an individual by analyzing genomic DNA isolated from peripheral blood nucleated cells or amniocytes is an alternative approach for determining the RBC antigen type. In this report, the allele specific polymerase chain reaction (AS-PCR) was used to identify the alleles at the MN and Ss loci that encode the corresponding antigens on glycophorin A (GPA) and glycophorin B (GPB), respectively. This method was used to type these alleles in peripheral blood samples obtained from normal individuals and from patients following massive transfusion. Of 23 peripheral blood specimens analyzed, all were correctly typed by this method. The allele specific polymerase chain reaction was also used to determine these alleles using amniotic fluid samples. Of 11 amniotic fluid specimens analyzed, 8 were correctly typed at both loci. Mistyping of three amniotic fluid specimens was explained by possible maternal blood contamination.

Stable secretion of a soluble, oligomeric form of rabies virus glycoprotein: influence of N-glycan processing on secretion.

Rabies virus glycoprotein (RGP) is a 505 amino acid type I transmembrane glycoprotein that is important in the pathogenesis of rabies virus infection. RGP also stimulates the development of neutralizing antibodies by the host. N-linked glycosylation is required for both cell surface expression and immunogenicity of RGP. In the current study, a soluble form of RGP, constructed by insertion of a stop codon external to the transmembrane domain, was expressed in transfected Chinese hamster ovary cells. The soluble form of RGP was found to be appropriately antigenic and immunogenic. Similar to full-length RGP, the soluble form was assembled into homodimers and homotrimers. Core glycosylation was required for secretion of soluble RGP and cell surface expression of full-length RGP. In addition, initial glucose trimming of the N-glycans was necessary and sufficient for secretion of soluble RGP and cell surface expression of full-length RGP. Further N-glycan processing was not required for secretion or cell surface expression of soluble or full-length RGP, respectively.

A molecular biologic approach to study the fine specificity of antibodies directed to the MN human blood group antigens.

The human MN blood group Ags on glycophorin A are linear complex glycopeptide Ags determined by a combination of amino acid polymorphisms and O-glycans. M Ag has Ser and Gly, and N Ag has Leu and Glu, at positions 1 and 5, respectively. Amino acids 2 to 4 are O-glycosylated. To analyze the fine specificity of Abs recognizing these Ags, recombinant glycophorin A molecules were expressed in Chinese hamster ovary (CHO) cells. The M-allele cDNA was used to generate the N-allele by site-directed mutagenesis. Two chimeric mutants were similarly constructed: Gly5-->Glu mimics the rare Mc phenotype; Ser1-->Leu is not found in human populations. Each type of glycophorin A was transfected into wild type CHO cells. In addition, the M-allele was expressed by mutant CHO cells defective in sialylation. The binding of M and N Abs and an anti-N lectin to recombinant glycophorin A was assessed by various methods. Two anti-N mouse mAbs and the anti-N lectin required leucine at position 1, whereas Glu5 was not essential. One anti-M mAb required both Gly5 and sialic acid. Three human anti-M sera required Ser1, whereas Gly5 was not essential. Four anti-M and -N mouse mAbs failed to bind recombinant glycophorin A, probably due to undersialylation of the recombinant glycoprotein. These results show that CHO cells expressing glycophorin A molecules varying in amino acid sequence and carbohydrate composition are useful for studying the fine specificity of Ab and lectin interactions with this glycoprotein. This is a novel approach and model system for investigating the immune response to linear complex glycopeptide Ags, a class of Ags that has received little attention previously.

Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A.

Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N-linked glycosylation at Asn26 of this glycoprotein by affecting the Asn-X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation-deficient Chinese hamster ovary (CHO) cells. When expressed in wild-type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O-linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N-acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N-acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N-linked glycosylation.

Efficiency of N-linked core glycosylation at asparagine-319 of rabies virus glycoprotein is altered by deletions C-terminal to the glycosylation sequon.

In N-linked core glycosylation, the oligosaccharide Glc3Man9GlcNAc2 is transferred to the tripeptide sequon Asn-X-Ser/Thr. However, this process must be regulated by additional protein signals, since many sequons are either poorly glycosylated or not glycosylated at all. Since N-linked glycosylation can influence protein structure and function, understanding these signals is essential for the design and expression of recombinant glycoproteins. Core glycosylation usually occurs cotranslationally in the rough endoplasmic reticulum (RER) during translocation of nascent proteins. Since only regions of a protein immediately near to a sequon or N-terminal to it are thought to be in the RER when core glycosylation occurs, most models predict that regions C-terminal to the sequon do not influence this process. We tested whether regions C-terminal to a sequon can influence its core glycosylation. Full-length (505 amino acid) rabies virus glycoprotein (RGP) mutants, each containing only one of the three sequons normally present in RGP, were used for these studies. Using a cell-free system, the core glycosylation efficiency at each sequon was determined. Termination codons were then introduced into these mutants at defined sites to produce C-terminal truncations, and the effect of each of these truncations on the core glycosylation efficiency at each sequon was assessed. While deletion of the C-terminal transmembrane and cytoplasmic domains did not affect core glycosylation, more extensive C-terminal deletions did result in altered core glycosylation in a site-specific fashion. Specifically, C-terminal truncations resulting in proteins containing 386 or 344 amino acids decreased the efficiency of core glycosylation at Asn319. This demonstrates that core glycosylation efficiency can be influenced by the presence or absence of regions in a protein more than 68 amino acids C-terminal to a specific glycosylation site.

N-linked glycosylation of beta-amyloid precursor protein.

The beta-amyloid peptide that accumulates in the brain of patients with Alzheimer's disease is derived by proteolytic processing of a family of membrane bound beta-amyloid precursor proteins (beta APPs). The three major isoforms of beta APP, derived by alternative splicing, contain 695, 751, and 770 amino acids. They are heavily O-glycosylated and contain two N-linked glycosylation sites. The pathways leading to beta-amyloid deposition in brain are not clear. It is possible that defects in metabolic and processing pathways of beta APP lead to the increased production and deposition of beta-amyloid. In many cases post-translational modifications, such as glycosylation, are important in regulating such pathways. We studied N-linked glycosylation of the 695 amino acid form of beta APP in detail by deleting the two potential glycosylation sites at Asn467 and Asn496. The mutants were examined both in a cell-free transcription/translation/glycosylation system and in transfected Chinese hamster ovary (CHO) cells. In both systems, only Asn467 was glycosylated. In CHO cells the N-linked oligosaccharide on beta APP was completely resistant to Endoglycosidase H, suggesting that it is of complex type. These mutants will be useful for studying the role of glycosylation in the metabolism of beta APP.

N-linked glycosylation of rabies virus glycoprotein. Individual sequons differ in their glycosylation efficiencies and influence on cell surface expression.

Many eukaryotic proteins are modified by N-linked glycosylation, a process in which oligosaccharides are added to asparagine residues in the sequon Asn-X-Ser/Thr. However, not all such sequons are glycosylated. For example, rabies virus glycoprotein (RGP) contains three sequons, only two of which appear to be glycosylated in virions. To examine further the signals in proteins which regulate N-linked core glycosylation, the glycosylation efficiencies of each of the three sequons in the antigenic domain of RGP were compared. For these studies, mutants were generated in which one or more sequons were deleted by site-directed mutagenesis. Core glycosylation of these mutants was studied using two independent systems: 1) in vitro translation in rabbit reticulocyte lysate supplemented with dog pancreatic microsomes, and 2) transfection into glycosylation-deficient Chinese hamster ovary cells. Parallel results were obtained with both systems, demonstrating that the sequon at Asn37 is inefficiently glycosylated, the sequons at Asn247 and Asn319 are efficiently glycosylated, and the glycosylation efficiency of each sequon is not influenced by glycosylation at other sequons in this protein. High levels of cell surface expression of RGP in Chinese hamster ovary cells are seen with any mutant containing an intact sequon at Asn247 or Asn319, whereas low levels of cell surface expression are seen when the sequon at Asn37 is present alone; deletion of all three sequons completely blocks RGP cell surface expression. Thus, although core glycosylation at Asn37 is inefficient, it is still sufficient to support a biological function, cell surface expression. Future studies using mutagenesis of this model protein and its expression in these two well defined systems will aim to begin to unravel the rules governing core glycosylation of glycoproteins.

Influence of duplexes 3' to the mRNA initiation codon on the efficiency of monosome formation.

The structural features of mRNA molecules that determine their relative translational rates are at present poorly defined. An early and potentially rate-limiting step in this process is the assembly of an intact 80S ribosome at the translational initiation codon. It is generally assumed that the efficiency of this reaction is controlled by structures in the 5' nontranslated region and in the immediate proximity of the AUG initiation codon. In this paper, we present an assay of initial monosome formation and measure the effects of hybridizing mRNA to complementary DNA fragments on the efficiency of this reaction. This hybridization serves to block specific regions of the mRNA from sequence-specific and intramolecular (secondary structure) interactions. We find that cDNAs that block the 5' nontranslated region, the initiation codon, or regions immediately 3' to the initiation codon markedly inhibit 80S ribosome attachment. These results are consistent with previous studies by ourselves and others which suggest that the introduction of secondary structures into this region can result in decreased translational efficiency. In addition, however, we note that cDNAs that hybridize to segments of the coding region significant distances (as many as several hundred bases) 3' to the initiation codon can also inhibit initial ribosome binding. This effect appears to be limited to duplexes within the mRNA coding region since a cDNA hybridizing exclusively within the 3' nontranslated region does not inhibit, and may actually stimulate, monosome formation. The results of this monosome formation assay therefore suggest that mRNA structures remote from the 5' terminus and initiation codon may also be important in determining the efficiency of translational initiation.