Sharon S. Krag

Proteomic Analysis of Chinese Hamster Ovary Cells

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life.

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.

Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a Trypanosoma brucei Glycosylation Mutant

ABSTRACT Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol-oligosaccharides might alter the degree of beta-1,6 branching in N-linked carbohydrates are discussed.

Physiologic and pathophysiologic consequences of altered sialylation and glycosylation on ion channel function

Voltage-gated ion channels are transmembrane proteins that regulate electrical excitability in cells and are essential components of the electrically active tissues of nerves, muscle and the heart. Potassium channels are one of the largest subfamilies of voltage sensitive channels and are among the most-studied of the voltage-gated ion channels. Voltage-gated channels can be glycosylated and changes in the glycosylation pattern can affect ion channel function, leading to neurological and neuromuscular disorders and congenital disorders of glycosylation (CDG). Alterations in glycosylation can also be acquired and appear to play a role in development and aging. Recent studies have focused on the impact of glycosylation and sialylation on ion channels, particularly for voltage-gated potassium and sodium channels. The terminal step of sialylation often affects channel activation and inactivation kinetics. The presence of sialic acids on O or N-glycans can alter the gating mechanism and cause conformational changes in the voltage-sensing domains due to sialic acids negative charges. This manuscript will provide an overview of sialic acids, potassium and sodium channel function, and the impact of sialylation on channel activation and deactivation.

Lectin-resistant CHO cells : selection of seven new mutants resistant to ricin

In attempts to isolate new CHO glycosylation mutants, selection protocols using plant lectins that bind galactose residues of cell surface carbohydrates were applied to mutagenized CHO populations. The lectins were used alone or in combination to obtain seven ricin-resistant phenotypes. Each mutant had distinctive properties compared with previously described ricin-resistant CHO cells. One of the new phenotypes was dominant in somatic cell hybrids, and the others were recessive. Complementation analyses between related lectinresistant (LecR) phenotypes indicated that each new isolate represented a novel genotype. Five of the mutants had properties typical of new CHO glycosylation mutants. The remaining two mutants were not readily categorized. Although they did not appear to be ricin-internalization or protein-synthesis mutants, they also did not display the marked alterations in sensitivity to several lectins of different sugar specificity expected for glycosylation mutants. The seven new LecR mutants described in these studies brings the total number of different LecR CHO mutants isolated by this and other laboratories to about 40. Criteria for identifying new LecR mutations in CHO cells are discussed.

Chapter 5 Mechanisms and Functional Role of Glycosylation in Membrane Protein Synthesis

Publisher Summary This chapter discusses the mechanisms and functional role of glycosylation in membrane protein synthesis. The lipid moiety of the glycosylation intermediates in bacterial glycan synthesis is known to be a phosphorylated polyisoprenol lipid (undecaprenyl phosphate). As dolichol—a family of lipids characterized by having 16–22 isoprene units with the α-isoprene unit saturated—is the abundant polyisoprenol lipid in mammalian tissues, even the earliest report of glycosylation intermediates in mammalian tissues implicated dolichyl phosphate as the lipid carrier. There are three general approaches used to determine the role of protein-bound carbohydrate. First, the properties of glycosylated and nonglycosylated forms of an isolated protein are examined. Second, the function and compartmentalization of a protein in an untreated cell can be compared to that in a cell treated with an inhibitor of glycosylation. Then, the function of a glycoprotein in a normal cell can be compared with that in a mutant cell defective in glycosylation. The synthesis of asparagine-linked glycoproteins involves a complex series of reactions catalyzed by soluble and membrane-associated enzymes in at least three cellular compartments. Dolichyl phosphate is clearly involved in glycosylation; it remains to be determined whether other polyisoprenoids are as well.

Preliminary characterization of a Chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity

A novel screening procedure was developed for isolating Chinese hamster ovary cell mutants altered in the early steps of the biosynthesis of asparagine-linked glycoproteins. This procedure identifies cells with low intracellular levels of two lysosomal hydrolases, beta-glucuronidase and alpha-iduronidase. One mutant cell line isolated in this way, CHB 11-1-3, has low intracellular levels of seven lysosomal enzymes as compared to wild-type cells. Although CHB 11-1-3 synthesizes mannosylphosphoryldolichol and [Man]5[NAcG1cNH2]2-P-P-lipid, it fails to utilize these lipid intermediates to make normal amounts of [Glc]3[Man]9[NAcG1cNH2]2P-P-lipid. As a consequence of this glycosylation defect, this mutant transfers oligosaccharides of a different structure than wild type to the lysosomal enzyme beta-hexosaminidase. In addition, it underglycosylates its proteins.

Glycoproteomic and glycomic databases

Protein glycosylation serves critical roles in the cellular and biological processes of many organisms. Aberrant glycosylation has been associated with many illnesses such as hereditary and chronic diseases like cancer, cardiovascular diseases, neurological disorders, and immunological disorders. Emerging mass spectrometry (MS) technologies that enable the high-throughput identification of glycoproteins and glycans have accelerated the analysis and made possible the creation of dynamic and expanding databases. Although glycosylation-related databases have been established by many laboratories and institutions, they are not yet widely known in the community. Our study reviews 15 different publicly available databases and identifies their key elements so that users can identify the most applicable platform for their analytical needs. These databases include biological information on the experimentally identified glycans and glycopeptides from various cells and organisms such as human, rat, mouse, fly and zebrafish. The features of these databases - 7 for glycoproteomic data, 6 for glycomic data, and 2 for glycan binding proteins are summarized including the enrichment techniques that are used for glycoproteome and glycan identification. Furthermore databases such as Unipep, GlycoFly, GlycoFish recently established by our group are introduced. The unique features of each database, such as the analytical methods used and bioinformatical tools available are summarized. This information will be a valuable resource for the glycobiology community as it presents the analytical methods and glycosylation related databases together in one compendium. It will also represent a step towards the desired long term goal of integrating the different databases of glycosylation in order to characterize and categorize glycoproteins and glycans better for biomedical research.

Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP‐mannose levels

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5°C instead of the normal growth temperature of 34°C in order to allow for the isolation of temperature‐sensitive lesions. This selection/screening procedure resulted in the isolation of MI5–4 cells, which had three‐ to five‐fold lower incorporation of [2‐3H]mannose into mannose 6‐phosphate, mannose 1‐phosphate, GDP‐mannose, oligosaccharide‐lipid, and glycoprotein at 41.5°C. We detected no difference in the qualitative pattern of mannose‐labeled lipid‐linked oligosaccharides compared to parental cells. MI5–4 cells synthesized dolichol. The defect of MI5–4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight‐ to nine‐fold lower in hexokinase activity in MI5–4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP‐mannose in MI5–4 cells was 70% of normal. The phenotype of MI5–4 was a lower specific activity of labeled GDP‐mannose, not a substantial reduction in the level of GDP‐mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low‐glucose medium. J. Cell. Biochem. 72:56–66, 1999.