Construction and analysis of a novel peptide tag containing an unnatural N-glycosylation site

The addition of N-glycans to clinically used proteins enhances their therapeutic features. Here we report the design of a novel peptide tag with an unnatural N-glycosylation site, which may increase the N-glycan content of generally any protein. The designed GlycoTags were attached to A1AT, EPO and AGP and constructs were expressed in HEK293 or CHO cells. Hereby we could prove that the attached unnatural N-glycosylation site is decorated with complex-type N-glycans and that the spacer as well as the C-terminal "tail" sequence are critical for the usage of the novel N-glycosylation site. This demonstrates that the novel GlycoTag is a convenient tool to provide proteins with extra N-glycan moieties by simply adding a peptide tag sequence as small as 22 amino acids.     

A Systems View of the Heparan Sulfate Interactome

Heparan sulfate proteoglycans consist of a small family of proteins decorated with one or more covalently attached heparan sulfate glycosaminoglycan chains. These chains have intricate structural patterns based on the position of sulfate groups and uronic acid epimers, which dictate their ability to engage a large repertoire of heparan sulfate–binding proteins, including extracellular matrix proteins, growth factors and morphogens, cytokines and chemokines, apolipoproteins and lipases, adhesion and growth factor receptors, and components of the complement and coagulation system. This review highlights recent progress in the characterization of the so-called “heparan sulfate interactome,” with a major focus on systems-wide strategies as a tool for discovery and characterization of this subproteome. In addition, we compiled all heparan sulfate–binding proteins reported in the literature to date and grouped them into a few major functional classes by applying a networking approach.     

Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction.

Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.     

Fatty acylation of cellular proteins. Temporal and subcellular differences between palmitate and myristate acylation

Previous studies demonstrated that palmitate and myristate are covalently linked to distinct sets of cellular proteins and that the linkages through which these fatty acids are attached to the polypeptide chains are different (Olson, E. N., Towler, D. A., and Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790). In the present study, the kinetics and subcellular sites of acylation of proteins with palmitate and myristate were examined in the BC3H1 muscle cell line. Acylation with myristate was an extremely early modification that appeared to take place cotranslationally or shortly thereafter for a variety of soluble and membrane-bound proteins. In contrast, acylation of proteins with palmitate was a post-translational event that occurred exclusively on membrane proteins. To begin to understand the intracellular pathways that acyl proteins follow during their maturation, the degree of glycosylation, and the nature of the interaction of these proteins with membranes were examined. The majority of acyl proteins were tightly associated with membranes and could not be removed by conditions that release peripheral proteins from membranes. However, only a minor fraction of acylated proteins were N-glycosylated. These data suggest that the acyltransferases that attach palmitate and myristate to proteins are present in different subcellular locations and demonstrate that these fatty acids are attached to newly synthesized acyl proteins at different times during their maturation. The lack of carbohydrate on the majority of integral membrane acyl proteins suggests that these proteins may follow intracellular pathways that are different from those followed by cell surface glycoproteins.     

The action of neutral hypochlorite on epithelial mucopolysaccharides

1. The uptake of dilute neutral hypochlorite by epithelial mucopolysaccharides has been compared with that of proteins, polysaccharides, amino acids and sugars. The uptake has been shown to be related to the protein content of the mucopolysaccharides rather than their polysaccharide content. 2. The destruction of the components of epithelial mucopolysaccharides, certain sugars and polysaccharides after oxidation with dilute neutral hypochlorite at 0-4 degrees has been studied. Very little destruction of the sugar components occurred and in epithelial mucopolysaccharides the only amino acid destroyed specifically was arginine. 3. Oxidation of bovine submaxillary-gland mucoprotein and ovalbumin caused very little destruction of hexosamine and no detectable liberation of this residue as a free reducing group, indicating that the O-seryl galactosaminide and the N-acyl-glycosylamine linkages reported to be present in these compounds were relatively stable to hypochlorite. 4. Depolymerization of epithelial mucopolysaccharides by neutral hypochlorite has been studied by using gel-filtration columns and compared with the depolymerization of polysaccharides and proteins under similar conditions. The polysaccharides examined were relatively resistant to oxidation whereas the proteins were extensively broken down. It is inferred that the extensive depolymerization of epithelial mucopolysaccharides by hypochlorite is related to their protein content rather than their polysaccharide content. 5. Fractionation of the products of oxidation of epithelial mucopolysaccharides by column procedures has revealed that the relative proportions of components in all fractions were similar to those in the original material. 6. Though this study does not provide unequivocal evidence from which the overall structure of this type of epithelial mucopolysaccharide may be deduced, the balance of probabilities now appears to favour a long polypeptide chain to which a large number of oligosaccharide side chains are attached via O-seryl and O-threonyl glycosidic linkages. The results, however, are also consistent with an alternating sequence of short polysaccharide and polypeptide chains and further evidence is necessary before this structure can be ruled out.     

Protein acylation in Tetrahymena

Examination of exhaustively delipidated Tetrahymena mimbres cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of several protein bands containing covalently linked fatty acids. Palmitic (16:0) and stearic (18:0) acids together accounted for approximately 90% of the protein-linked acyl chains, with myristic acid (14:0) comprising most of the remainder. Each of these three fatty acids was present mainly in alkali-stable linkage, indicating that unlike most other systems examined, fatty acids are attached to proteins of Tetrahymena principally by amide bonds. Smaller proportions of the acyl chains were susceptible to release by hydroxylaminolysis or by alkaline hydrolysis as would be expected from an ester linkage. The protein-bound acyl chains accounted for 0.3% of the cells' total fatty acids. They closely resembled in composition the highly saturated free fatty acid pool but not the vast pool of glycerolipid-associated fatty acids, which were mainly unsaturated. Cells subjected to thermal stress by rapid chilling from 39 to 15 degrees C responded by sharply increasing the ratio of palmitate to stearate in covalent association with proteins.     

Protein acylation in normoxic and ischemic/reperfused cardiac tissue.

In addition to a prominent role in tissue energy conversion, fatty acids are involved in signal transduction and modulation of cellular protein localization and function. The latter is accomplished by acylation of specific cellular proteins. In the present study the amount of fatty acyl moieties covalently bound to cardiac proteins and the effect of myocardial ischemia and reperfusion on the degree and relative fatty acyl composition of cardiac proteins have been investigated in isolated rat hearts. In the normoxic heart about 0.32% of the cellular fatty acyl pool is covalently bound to proteins. Approximately 90% of these fatty acyl chains are thio-esterified, whereas a relatively minor part is attached to cardiac proteins through amide linkage. Thio-esterified fatty acyl chains are derived from palmitic, stearic, oleic, linoleic, arachidonic and docosahexaenoic acid. In contrast, amide linked protein acylation shows a preference for myristic acyl chains. Acute ischemia and reperfusion inflicted upon the isolated rat heart did enhance significantly the content of (unesterified) fatty acids, but did neither affect the degree of protein acylation nor the relative fatty acyl composition of acylated proteins in cardiac tissue.     

Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells

The asparagine-linked sugar chains of natural interferon-beta 1 secreted from human foreskin fibroblasts by poly I:poly C induction and of three recombinant human interferon-beta 1 produced by Chinese hamster ovary cells, mouse epithelial cells (C127), and human lung adenocarcinoma cells (PC8) were released quantitatively as oligosaccharides by hydrazinolysis followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were comparatively analyzed. More than 80% of the sugar chains of natural interferon-beta 1 occur as biantennary complex-type sugar chains, approximately 10% of which contain N-acetyllactosamine repeating structure in their outer chain moieties. The remainders are 2,4- and 2,6-branched triantennary complex-type sugar chains. The sugar chains of the recombinant interferon-beta 1 derived from Chinese hamster ovary cells were very similar to those of its natural counterpart. In contrast, two other recombinant proteins contain quite different sugar chains. The protein derived from C127 cells contains complex-type sugar chains with the Gal alpha 1----3Gal beta 1----4GlcNAc group in their outer chain moieties. Their sialic acid residues occur solely as the Sia alpha 2----6Gal group, where Sia is sialic acid. In contrast, the sialic acid residues of other interferon-beta 1 occur as the Sia alpha 2----3Gal group only. A part of the sugar chains of the protein derived from PC8 cells contains bisecting N-acetylglucosamine residue in addition to the Gal alpha 1----3Gal beta 1----4GlcNAc group.     

Structural remodeling, trafficking and functions of glycosylphosphatidylinositol-anchored proteins

Glycosylphosphatidylinositol (GPI) is a glycolipid that is covalently attached to proteins as a post-translational modification. Such modification leads to the anchoring of the protein to the outer leaflet of the plasma membrane. Proteins that are decorated with GPIs have unique properties in terms of their physical nature. In particular, these proteins tend to accumulate in lipid rafts, which are critical for the functions and trafficking of GPI-anchored proteins (GPI-APs). Recent studies mainly using mutant cells revealed that various structural remodeling reactions occur to GPIs present in GPI-APs as they are transported from the endoplasmic reticulum to the cell surface. This review examines the recent progress describing the mechanisms of structural remodeling of mammalian GPI-anchors, such as inositol deacylation, glycan remodeling and fatty acid remodeling, with particular focus on their trafficking and functions, as well as the pathogenesis involving GPI-APs and their deficiency.     

The nature of the protein moieties of cartilage proteoglycans of pig and ox.

Proteoglycans extracted with 4M-guanidinium chloride from pig laryngeal cartilage and bovine nasal septum were purified by density-gradient centrifugation in CsCl under 'associative' followed by 'dissociative' conditions [Hascall & Sajdera (1969) J. Biol. Chem. 244, 2384-2396]. Proteoglycans were then digested exhaustively with testicular hyaluronidase, which removed about 80% of the chondroitin sulphate. The hyaluronidase was purified until no proteolytic activity was detectable under the conditions used for digestion. The resulting 'core' proteins of both species were fractionated by a sequence of gel-chromatographic procedures which gave four major fractions of decreasing hydrodynamic size. Those that on electrophoresis penetrated 5.6% (w/v) polyacrylamide gels migrated as discrete bands whose mobility increased with decreasing hydrodynamic size. The unfractionated 'core' proteins had the same N-terminal amino acids as the intact proteoglycan, suggesting that no peptide bonds had been cleaved during hyaluronidase digestion. Alanine predominated as the N-terminal residue in all the fractions of both species. Fractions were analysed for amino acid, amino sugar, uronic acid and neutral sugar compositions. In pig 'core' proteins, the glutamic acid content increased significantly with hydrodynamic size, but in bovine 'core' proteins this trend was less marked. Significant differences in amino acid composition between fractions suggested that in each species there was more than one variety of proteoglycan. The molar proportions of xylose to serine destroyed on alkaline beta-elimination were equivalent in most fractions, indicating that the serine residues destroyed were attached to the terminal xylose of chondroitin sulphate chains. The ratio of serine residues to threonine residues destroyed on beta-elimination, was similar in all fractions of both species. Since the fractions of smallest hydrodynamic size contained less keratan sulphate than those of larger size, it implies that in the former the keratan sulphate chains were shorter than in the latter.     

Expression of human glycosyltransferase genes in yeast as a tool for enzymatic synthesis of sugar chain

We planned the production of human glycosyltransferases in yeast for the enzymatic synthesis of various sugar chains. More than 160 genes encoding various glycosyltransferases were prepared as N-terminal transmembrane region truncated forms by PCR and were inserted into the entry vector of Invitrogen Ltd's Gateway system. About fifty glycosyltransferases were chosen for the synthesis of human type oligosaccharides, and expressed as two different forms in yeast. One is a soluble form, which is secreted into the culture medium by methylotrophic yeast, and the other is an immobilized form, which is displayed at the budding yeast cell wall as a fusion protein with Pir protein. To date, in both systems, some sialyltranferases and fucosyltransferases have been produced as active forms, indicating the potential usefulness of these systems for the enzymatic synthesis of various types of human sugar chains attached to proteins and lipids.     

Biochemical characterization and biosynthesis of the uterine milk proteins of the pregnant sheep uterus

The uterine milk proteins (UTM-proteins), a pair of basic glycoproteins with similar isoelectric points and molecular weights (57,000 and 55,000), are secreted by the endometrium of the pregnant ewe. Peptide mapping of the two species of UTM-proteins demonstrated them to be structurally related. Furthermore, pulse-chase and continuous-labeling experiments indicated that both are produced from a common precursor of lower molecular weight. Purified UTM-proteins were found to be rich in basic amino acids, low in tyrosine, and apparently lacking in tryptophan. The proteins were about 5.6-5.7% carbohydrate by weight and bound the lectin, concanavalin A. UTM-proteins synthesized in vitro incorporated D-[3H]glucosamine. Analysis of [3H]glucosamine-labeled glycopeptides of Pronase-digested UTM-proteins by gel filtration indicated that most radioactivity is associated with one size class of oligosaccharide. UTM-proteins secreted by the endometrium in the presence of tunicamycin, an N-glycosylation inhibitor, were of lower molecular weight than those from control endometria, indicating that sugar chains are attached to the protein core via N-linkages to asparagine. UTM-proteins synthesized in culture incorporated [32P]orthophosphate, and tunicamycin inhibited this incorporation. Analysis of hydrolyzed UTM-proteins by paper chromatography indicated that much of the 32P was associated with mannose 6-phosphate. Because this moiety is the so-called lysosomal recognition marker and is present on uteroferrin, the acid phosphatase of porcine uterine secretions, we tested UTM-proteins for several enzymatic activities characteristic of lysosomes, but none was found. In conclusion, the UTM-proteins are related glycoproteins that, like porcine uteroferrin, contain mannose 6-phosphate, a result which suggests that secretion of glycoproteins with phosphorylated oligosaccharide chains may be a common feature of the progestational uterus.     

Galectins as modulators of receptor tyrosine kinases signaling in health and disease

Receptor tyrosine kinases (RTKs) constitute a large group of cell surface proteins that mediate communication of cells with extracellular environment. RTKs recognize external signals and transfer information to the cell interior, modulating key cellular activities, like metabolism, proliferation, motility, or death. To ensure balanced stream of signals the activity of RTKs is tightly regulated by numerous mechanisms, including receptor expression and degradation, ligand specificity and availability, engagement of co-receptors, cellular trafficking of the receptors or their post-translational modifications. One of the most widespread post-translational modifications of RTKs is glycosylation of their extracellular domains. The sugar chains attached to RTKs form a new layer of information, so called glyco-code that is read by galectins, carbohydrate binding proteins. Galectins are family of fifteen lectins implicated in immune response, inflammation, cell division, motility and death. The versatility of cellular activities attributed to galectins is a result of their high abundance and diversity of their cellular targets. A various sugar specificity of galectins and the differential ability of galectin family members to form oligomers affect the spatial distribution and the function of their cellular targets. Importantly, galectins and RTKs are tightly linked to the development, progression and metastasis of various cancers. A growing number of studies points on the close cooperation between RTKs and galectins in eliciting specific cellular responses. This review focuses on the identified complexes between galectins and RTK members and discusses their relevance for the cell physiology both in healthy tissues and in cancer.     

Determination of glycosylation sites, disulfide bridges, and the C-terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry.

Stereum purpureum endopolygalacturonase (endoPG; EC 3.2.1.15) is a causal protein for silver-leaf disease in apple trees. Endopolygalacturonase I, is a mixture of three components (Ia, Ib, and Ic) that produce three bands on SDS/PAGE but have the same polypeptide and sugar chains. Electrospray ionization mass spectrometry (ESI-MS) analysis of three endoPG I proteins and deglycosylated endoPG Ia revealed a molecular mass of 37 068, 38 285, and 39 503 for Ia, Ib, and Ic, respectively; the number of N-binding sugar chains matches that of a high-mannose type of sugar chain. Two, three, and four sugar chains are present in endoPG Ia, Ib, and Ic, respectively. Deletion of 44 amino acids from the deduced sequence occurred in the C-terminal region. Positions of the glycosylation sites and disulfide bridges were decided by tryptic digestion followed by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) analysis of reductive and nonreductive pyridylethylated endoPG I proteins. The glycosylated asparagines were determined to be Asn92 and 161; Asn92, 161, 279, or 302; and Asn92, 161, 279, and 302 in Ia, Ib, and Ic, respectively. Three disulfide bridges were noted at Cys3-Cys17, Cys175-Cys191, and Cys300-Cys303. These results are the first findings for fungal endoPG and may contribute to clarification of the relationship between stereostructure and catalytic activity.     

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.     

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p -chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide- m -chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the 'empty seed coat technique'. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier-mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy-dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half-times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half-times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.     

k Chain variable regions from three galactan binding myeloma proteins.

A series of seven BALB/c myeloma proteins has been identified with binding specificity for antigens containing beta(1 leads to 6)-D-galactopyranosyl moieties. We have determined the primary amino acid sequence of the first 108 residues from the light chains of three of these proteins. The framework portions of the variable regions of these three light chains are identical with residue 100 at which position three different amino acids are found in the three chains. An additional interchange was found at position 106 in one of the proteins. Based on recent DNA sequence studies suggesting that the variable region ends at residue 97, these substitutions indicate the possible existance of multiple genes coding for the region beginning at residue 98 and continuing toward the carboxy terminus. A single amino acid interchange was observed in complementarity determining regions occurring in L3. This substitution (Ile-Trp) would require changes in all three codon bases to produce the respective amino acids if one were derived from the other. Two of these chains are thus indistinguishable for their first 100 amino acids and are the first pair of k chains to exhibit complete identity over their variable regions.     

“Lost sugars” — reality of their biological and medical applications

The glycan chains attached to cell surfaces or to single proteins are highly dynamic structures with various functions. The glycan chains of mammals and of some microorganisms often terminate in sialic acids or α-1,3-galactose. Although these two sugars are completely distinct, there are several similarities in their biological and medical importance. First, one type of sialic acid, N-glycolylneuraminic acid, and the galactose bound by an α-1,3-linkage to LacNAc, that forms an α-gal epitope, were both eliminated in human evolution, resulting in the production of antibodies to these sugars. Both of these evolutionary events have consequences connected with the consumption of foods of mammalian origin, causing medical complications of varying severity. In terms of ageing, sialic acids prevent the clearance of glycoproteins and circulating blood cells, whereas cryptic α-gal epitopes on senescent red blood cells contribute to their removal from circulation. The efficiency of therapeutic proteins can be increased by sialylation. Another common feature is the connection with microorganisms since sialic acids and α-gal epitopes serve as receptors on host cells and can also be expressed on the surfaces of some microorganisms. Whereas, the sialylation of IgG antibodies may help to treat inflammation, the expression of the α-gal epitope on microbial antigens increases the immunogenicity of the corresponding vaccines. Finally, sialic acids and the α-gal epitope have applications in cancer immunotherapy. N-glycolylneuraminic acid is a powerful target for cancer immunotherapy, and the α-gal epitope increases the efficiency of cancer vaccines. The final section of this article contains a brief overview of the methods for oligosaccharide chain synthesis and the characteristics of sialyltransferases and α-1,3-galactosyltransferase.     

Assessment of in vitro binding of isolated pectic domains to cellulose by adsorption isotherms, electron microscopy, and X-ray diffraction methods

Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic domains responsible for binding with cellulose microfibrils.