Proteome-wide characterization of N-glycosylation events by diagonal chromatography

A procedure to map N-glycosylation sites is presented here. It can be applied to purified proteins as well as to highly complex mixtures. The method exploits deglycosylation by PNGase F in a diagonal, reverse-phase chromatographic setup. When applied to 10 microL of mouse serum, affinity-depleted for its three most abundant components, 117 known or predicted sites were mapped in addition to 10 novel sites. Several sites were detected on soluble membrane or receptor components. Our method furthermore senses the nature of glycan structures and can detect differential glycosylation on a given site. These properties--high sensitivity and dependence on glycan imprinting--can be exploited for glycan-biomarker analysis.     

N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule

Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn(37), Asn(247), and Asn(319), although Asn(37) is not efficiently glycosylated. To examine N-glycan processing at Asn(247) and Asn(319), full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn(247) sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn(319) in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn(247) and Asn(319). However, Asn(247) had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn(319). Moreover, when comparing the N-glycans at Asn(319) on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn(247) allows additional N-glycan processing to occur at Asn(319), yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined.     

Selective control of oligosaccharide transfer efficiency for the N-glycosylation sequon by a point mutation in oligosaccharyltransferase

Asn-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria. Oligosaccharyltransferase (OST) catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. Inefficient oligosaccharide transfer results in glycoprotein heterogeneity, which is particularly bothersome in pharmaceutical glycoprotein production. Amino acid variation at the X position of the Asn-X-Ser/Thr sequon is known to modulate the glycosylation efficiency. The best amino acid at X is valine, for an archaeal Pyrococcus furiosus OST. We performed a systematic alanine mutagenesis study of the archaeal OST to identify the essential and dispensable amino acid residues in the three catalytic motifs. We then investigated the effects of the dispensable mutations on the amino acid preference in the N-glycosylation sequon. One residue position was found to selectively affect the amino acid preference at the X position. This residue is located within the recently identified DXXKXXX(M/I) motif, suggesting the involvement of this motif in N-glycosylation sequon recognition. In applications, mutations at this position may facilitate the design of OST variants adapted to particular N-glycosylation sites to reduce the heterogeneity of glycan occupancy. In fact, a mutation at this position led to 9-fold higher activity relative to the wild-type enzyme, toward a peptide containing arginine at X in place of valine. This mutational approach is potentially applicable to eukaryotic and eubacterial OSTs for the production of homogenous glycoproteins in engineered mammalian and Escherichia coli cells.     

Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

ABSTRACT

The placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2.     

Proteome-wide mapping of short-lived proteins in human cells

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Whole-genome linkage analysis in mapping alcoholism genes using single-nucleotide polymorphisms and microsatellites

There is currently a great interest in using single-nucleotide polymorphisms (SNPs) in genetic linkage and association studies because of the abundance of SNPs as well as the availability of high-throughput genotyping technologies. In this study, we compared the performance of whole-genome scans using SNPs with microsatellites on 143 pedigrees from the Collaborative Studies on Genetics of Alcoholism provided by Genetic Analysis Workshop 14. A total of 315 microsatellites and 10,081 SNPs from Affymetrix on 22 autosomal chromosomes were used in our analyses. We found that the results from the two scans had good overall concordance. One region on chromosome 2 and two regions on chromosome 7 showed significant linkage signals (i.e., NPL >or= 2) for alcoholism from both the SNP and microsatellite scans. The different results observed between the two scans may be explained by the difference observed in information content between the SNPs and the microsatellites.     

Multiplex detection of single-nucleotide variations using molecular beacons

We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surrounding the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly complementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, we prepared four template DNAs that only differed from one another by the identity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated with only one of the four template DNAs. The results show that in each reaction a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecular beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amplification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beacons. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assays to be carried out for genetic analysis.     

Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis

N^ɛ-Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of N^ɛ-lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. Twenty lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in the persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence.     

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.     

Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants.

We have compared the site-by-site N-glycosylation status of human lactoferrin (Lf) produced in maize, a monocotyledon, and in tobacco, used as a model dicotyledon. Maize and tobacco plants were stably transformed and recombinant Lf was purified from both seeds and leaves. N-glycopeptides were generated by trypsin digestion of recombinant Lf and purified by reverse-phase HPLC. The N-glycosylation pattern of each site was determined by mass spectrometry. Our results indicated that the N-glycosylation patterns of recombinant Lf produced in maize and tobacco share common structural features. In particular, both N-glycosylation sites of each recombinant Lf are mainly substituted by typical plant paucimannose-type N-glycans, with beta1,2-xylose and alpha1,3-linked fucose at the proximal N-acetylglucosamine. However, tobacco Lf shows a significant amount of processed N-glycans with one or two beta1,2GlcNAc linked to the trimannose core, which are weakly expressed in maize Lf. Finally, no Lewisa epitope was observed on tobacco Lf.     

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium

The archaeon Halobacterium halobium expresses a cell surface glycoprotein (CSG) with a repeating pentasaccharide unit N- glycosidically linked via N-acetylgalactosamine to Asn-2 of the polypeptide (GalNAc(1-N)Asn linkage type). This aspar-agine of the linkage unit is located within the N-terminal sequence Ala-Asn-Ala-Ser- , in accordance with the tripeptide consensus sequence Asn-Xaa-Ser/Thr typical for nearly every N-glycosylation site known so far, which are of the GlcNAc(1-N)-Asn linkage type. By a gene replacement method csg mutants were created which replace the serine residue of the consensus sequence by valine, leucine, and asparagine. Unexpectedly, this elimination of the consensus sequence did not prevent N-glycosylation. All respective mutant cell surface glycoproteins were N-glycosylated at Asn-2 with the same N-glycan chain as the wild type CSG. Asn-479 is N- glyco-sylated via a Glc(1-N)Asn linkage type in the wild type CSG. Replacement of Ser-481 in the sequence Asn-Ser-Ser for valine prevented glycosylation of Asn-479. From these results we postulate the existence of two different N-glycosyltransferases in H.halobium, one of which does not use the typical consensus sequence Asn-Xaa-Ser/Thr necessary for all other N-glycosyltransferases described so far.      

Isolation of a glycosylated form of the chicken eggshell protein ovocleidin and determination of the glycosylation site. Alternative glycosylation/phosphorylation at an N-glycosylation sequon

Ovocleidin, a major protein of the avian eggshell calcified layer, occurs in the eggshell soluble organic matrix in at least two forms. The major form is a phosphoprotein with two phosphorylated serines (OC-17) which was sequenced recently. A minor form is a glycosylated protein with identical sequence and only one phosphorylated serine (OC-23). The site of glycosylation is Asn(59), the only asparagine in the amino acid sequence contained in the N-glycosylation site consensus sequence, N-A-S. Ser(61), which is part of this site, is phosphorylated in OC-17 but not in OC-23 indicating that the two modifications are mutually exclusive. This is the first example of alternative glycosylation/phosphorylation occurring at an N-glycosylation site.     

Use of denaturing HPLC to map human and murine genes and to validate single-nucleotide polymorphisms

Linkage mapping has been extensively applied in the murine and human genomes. It remains a powerful approach to mapping genes and identifying genetic variants. As genome efforts identify large numbers of single-nucleotide polymorphisms, it will be critical to validate these polymorphisms and confirm their gene assignment and chromosomal location. The presence of pseudogenes can confuse such efforts. We have used denaturing HPLC to identify polymorphisms in human genes and to genotype individuals in selected CEPH pedigrees. The same approach has been applied to the mapping of murine genes in interspecies backcross animals. This strategy is rapid, accurate and superior in several respects to other technologies.     

High-density single-nucleotide polymorphism maps of the human genome

Here we report a large, extensively characterized set of single-nucleotide polymorphisms (SNPs) covering the human genome. We determined the allele frequencies of 55,018 SNPs in African Americans, Asians (Japanese-Chinese), and European Americans as part of The SNP Consortium's Allele Frequency Project. A subset of 8333 SNPs was also characterized in Koreans. Because these SNPs were ascertained in the same way, the data set is particularly useful for modeling. Our results document that much genetic variation is shared among populations. For autosomes, some 44% of these SNPs have a minor allele frequency > or =10% in each population, and the average allele frequency differences between populations with different continental origins are less than 19%. However, the several percentage point allele frequency differences among the closely related Korean, Japanese, and Chinese populations suggest caution in using mixtures of well-established populations for case-control genetic studies of complex traits. We estimate that approximately 7% of these SNPs are private SNPs with minor allele frequencies <1%. A useful set of characterized SNPs with large allele frequency differences between populations (>60%) can be used for admixture studies. High-density maps of high-quality, characterized SNPs produced by this project are freely available.     

Structural requirements for protein N-glycosylation. Influence of acceptor peptides on cotranslational glycosylation of yeast invertase and site-directed mutagenesis around a sequon sequence

To understand better the structural requirements of the protein moiety important for N-glycosylation, we have examined the influence of proline residues with respect to their position around the consensus sequence (or sequon) Asn-Xaa-Ser/Thr. In the first part of the paper, experiments are described using a cell-free translation/glycosylation system from reticulocytes supplemented with dog pancreas microsomes to test the ability of potential acceptor peptides to interfere with glycosylation of nascent yeast invertase chains. It was found that peptides, being acceptors for oligosaccharide transferase in vitro, inhibit cotranslational glycosylation, whereas nonacceptors have no effect. Acceptor peptides do not abolish translocation of nascent chains into the endoplasmic reticulum. Results obtained with proline-containing peptides are compatible with the notion that a proline residue in an N-terminal position of a potential glycosylation site does not interfere with glycosylation, whereas in the position Xaa or at the C-terminal of the sequon, proline prevents and does not favour oligosaccharide transfer, respectively. This statement was further substantiated by in vivo studies using site-directed mutagenesis to introduce a proline residue at the C-terminal of a selected glycosylation site of invertase. Expression of this mutation in three different systems, in yeast cells, frog oocytes and by cell-free translation/glycosylation in reticulocytes supplemented with dog pancreas microsomes, leads to an inhibition of glycosylation with both qualitative and quantitative differences. This may indicate that host specific factors also contribute to glycosylation.     

Use of phage display for detecting single-nucleotide differences in genes]

Experimental strategy has been developed for selection of mismatched DNA binding phages from library of E. coli f1 filamentous phages carrying random peptide inserts on the surface of bacteriophage particles. The strategy is based on the use of phage display technique, DNA heteroduplexes (with single nucleotide variations), and paramagnetic beads. DNA heteroduplexes have been obtained from biotin-labeled PCR product. During the first stage the phage particles were incubated with DNA heteroduplexes possessing mismatched nucleotides. The next step after elimination of free phages and separation of bound phages from DNA heteroduplexes was subtraction of phages binding with DNA heteroduplexes (without mismatched nucleotides). Phages selected by this method were capable of discriminating DNA heteroduplexes with single nucleotide variations from DNA homoduplexes. Phages immobilized on solid base retain their activity and specificity, and therefore can be used for developing a new screening automated method for detecting point mutations and gene polymorphism.