The effects of limited proteolysis by trypsin on human haptoglobin

Trypsin digestion of haptoglobin resulted i four glycopeptides. The glycopeptides were characterized by amino acid composition and molecular weight, as determined by thin-layer chromatography, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. Hemoglobin-binding capacity and immunological properties were investigated. glycopeptides I and II did not form an active complex with hemoglobin and they inhibited the reaction of haptoglobin with specific antiserum by over 70%. Glycopeptides III and IV showed 11 and 4% of the hemoglobin-binding capacity and 82 and 67% of antigenic reactivity of native haptoglobin, respectively. Glycopeptide IV contained three antigenic determinants, whereas glycopeptides III contained four, one of them being exposed by trypsin digestion. In crossed two-dimensional immunoelectrophoresis, glycopeptide III showed at least four components reacting with antihaptoglobin serum, and glycopeptide IV, two components.     

Studies on haptoglobin binding to concanavalin A

Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.     

Attachment Sites of Carbohydrate Portions to Peptide Chain of κ-Casein from Bovine Colostrum

Short glycopeptides were prepared from bovine colostral κ-casein treated with cyanogen bromide and proteases (pronase P and thermolysin), followed by gel filtrations and ion exchange chromatography. It was confirmed by Edman degradation that glycopeptide I among short glycopeptides obtained was homogeneous. From the effect of alkali treatment, it was assumed that three polysaccharide chains of glycopeptide I were attached to the peptide chain through OH groups of threonines. By chemical procedures and carboxypeptidase P treatment, the amino acid sequence of glycopeptide I was established to be Ser-Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu-Ala-Val. Threonine residues of No. 5, 7 and 9 were bound to the carbohydrate chains through galactosamine. The sugar chain bound to the threonine residue at No. 7 contained glucosamine. Glycopeptide I corresponded to residues of No. 127–139 in κ-casein A from normal milk.     

Structure of carbohydrate chains of a high-molecular-weight pulmonary glycoprotein

A human, alveolar glycoprotein having an apparent mol. wt. of 250 000 gave two major glycopeptide fractions (I and II) by Pronase digestion, followed by gel filtration, DEAE-cellulose column chromatography, paper chromatography, and paper electrophoresis. Glycopeptide I contained d-galactose, d-mannose, 2-acetamido-2-deoxy-d-glucose, and N-acetylneuraminic acid in the molar ratio of 2:3:4:1, whereas these sugars were present in Glycopeptide II in the molar ratio of 2:3:4:2.l-Fucose was present only in Glycopeptide II at a concentration of one l-fucose per three d-mannose residues. In both glycopeptides, 2-acetamido-2-deoxy-d-glucose was linked to an asparagine residue of the peptide chain. Based on the results of alkaline borohydride treatment, periodate oxidation, methlylation analysis, and sequential glycosidase degradation of the glycopeptides, tentative structures are proposed for both glycopeptides.     

Haptoglobin biosynthesis in rats Immunological identification of polysomes synthesizing haptoglobin and quantitation of haptoglobin in the cytoplasm of liver cells

A quantitative enzyme-linked immunosorbent assay was developed and utilized to study the stimulation of haptoglobin biosynthesis during an acute inflammatory challenge. A 10-fold increase in intracellular haptoglobin was measured at the peak of the inflammatory response. The increase in serum haptoglobin levels was concomitant with the intracellular levels, demonstrating the secretory output is also elevated during the inflammatory period. A monospecific antihaptoglobin was produced and used to detect the specific polysomes involved in haptoglobin synthesis. The amount of radioactively labeled antibody bound to the nascent haptoglobin chain was increased approx. 3-fold during the inflammatory response, indicating that new haptoglobin was being synthesized and suggesting an increase in functional haptoglobin mRNA resulting from the inflammatory signal.     

Comparison of glycoproteins by two-dimensional mapping of glycosylated peptides

We describe here a two-dimensional mapping procedure which is capable of resolving glycopeptides isolated by lectin affinity chromatography from radioiodinated tryptic digests of glycoproteins. Glycopeptide maps were successfully produced for the model proteins alpha 1-acid glycoprotein and fetuin, as well as for the two surface glycoproteins gp90 and gp45 from equine infectious anemia virus (EIAV). Differences were detected in the glycopeptide maps obtained for the gp90 and gp45 components from two antigenically distinct strains of EIAV, demonstrating the ability of this procedure to detect variations in glycosylation in closely related glycoproteins. Thus this glycopeptide mapping technique provides a simple, rapid method to study changes in glycopeptides requiring only micrograms of glycoprotein.     

Structure of glycoproteins of human erythrocytes. Alkali-stable oligosaccharides

Studies have been made on the oligosaccharide residues of the alkali-stable carbohydrate-protein linkage of sialoglycopeptides derived from human erythrocytes. Four glycopeptides were isolated after alkaline borohydride treatment and Pronase digestion of MN-active sialoglycopeptides. The structure of one of these glycopeptides (GPIV) has been studied by sequential hydrolysis with specific glycosidases. Glycopeptide GPIV contained (per mol): 1mol of fucose, 1mol of sialic acid, 3mol of galactose, 3mol of mannose, 4mol of acetylglucosamine, 1mol of aspartic acid and fractional amounts of threonine, serine and glycine. The molecular weight of the glycopeptide was estimated to be 2330 by gel filtration. On the basis of glycosidase-digestion results, a tentative structure is proposed for the oligosaccharide moiety of glycopeptide GPIV.     

Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.     

Old and new glycopeptide antibiotics: From product to gene and back in the post-genomic era

Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by multi-drug resistant Gram-positive pathogens. First-generation glycopeptides (vancomycin and teicoplanin) are produced by soil-dwelling actinomycetes. Second-generation glycopeptides (dalbavancin, oritavancin, and telavancin) are semi-synthetic derivatives of the progenitor natural products. Herein, we cover past and present biotechnological approaches for searching for and producing old and new glycopeptide antibiotics. We review the strategies adopted to increase microbial production (from classical strain improvement to rational genetic engineering), and the recent progress in genome mining, chemoenzymatic derivatization, and combinatorial biosynthesis for expanding glycopeptide chemical diversity and tackling the never-ceasing evolution of antibiotic resistance.     

Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways.     

Glycopeptides of the Type-Common Glycoprotein gD of Herpes Simplex Virus Types 1 and 2

We have carried out detailed structural studies of the glycopeptides of glycoprotein gD of herpes simplex virus types 1 and 2. We first examined and compared the number of N-asparagine-linked oligosaccharides present in each glycoprotein. We found that treatment of either pgD-1 or pgD-2 with endo-β-N-acetylglucosaminidase H (Endo H) generated three polypeptides which migrated more rapidly than pgD on gradient sodium dodecyl sulfate-polyacrylamide gels. Two of the faster-migrating polypeptides were labeled with [³H]mannose, suggesting that both pgD-1 and pgD-2 contained three N-asparagine-linked oligosaccharides. Second, we characterized the [³H]mannose-labeled tryptic peptides of pgD-1 and pgD-2. We found that both glycoproteins contained three tryptic glycopeptides, termed glycopeptides 1, 2, and 3. Gel filtration studies indicated that the molecular weights of these three peptides were approximately 10,000, 3,900, and 1,800, respectively, for both pgD-1 and pgD-2. Three methods were employed to determine the size of the attached oligosaccharides. First, the [³H]mannose-labeled glycopeptides were treated with Endo H, and the released oligosaccharide was chromatographed on Bio-Gel P6. The size of this molecule was estimated to be approximately 1,200 daltons. Second, Endo H treatment of [³⁵S]methionine-labeled glycopeptide 2 reduced the molecular size of this peptide from approximately 3,900 to approximately 2,400 daltons. Third, glycopeptide 2 isolated from the gD-like molecule formed in the presence of tunicamycin was approximately 2,200 daltons. From these experiments, the size of each N-asparagine-linked oligosaccharide was estimated to be approximately 1,400 to 1,600 daltons. Our experiments indicated that glycopeptides 2 and 3 each contained one N-asparagine-linked oligosaccharide chain. Although glycopeptide 1 was large enough to accommodate more than one oligosaccharide chain, the experiments with Endo H treatment of the glycoprotein indicated that there were only three N-asparagine-linked oligosaccharides present in pgD-1 and pgD-2. Further studies of the tryptic glycopeptides by reverse-phase high-performance liquid chromatography indicated that all of the glycopeptides were hydrophobic in nature. In the case of glycopeptide 2, we observed that when the carbohydrate was not present, the hydrophobicity of the peptide increased. The properties of the tryptic glycopeptides of pgD-1 were compared with the properties predicted from the deduced amino acid sequence of gD-1. The size and amino acid composition compared favorably for glycopeptides 1 and 2. Glycopeptide 3 appeared to be somewhat smaller than would be predicted from the deduced sequence of gD-1. It appears that all three potential glycosylation sites predicted by the amino acid sequence are utilized in gD-1 and that a similar number of glycosylation sites are present in gD-2.     

Understanding and manipulating glycopeptide pathways: the example of the dalbavancin precursor A40926

Glycopeptide antibiotics represent an important class of microbial compounds produced by several genera of actinomycetes. The emergence of resistance to glycopeptides among enterococci and staphylococci has prompted the search for second-generation drugs of this class and semi-synthetic derivatives are currently under clinical trials. Dalbavancin is obtained by chemical modification of the natural glycopeptide A40926, produced by a Nonomuraea sp. Recently, there has been considerable progress in the elucidation of biosynthesis of glycopeptide antibiotics; several gene clusters have been characterized, thus providing an understanding of the biosynthesis of these chemically complex molecules. Furthermore, such investigations have yielded the first glycopeptide derivatives produced by genetic or enzymatic intervention. We have isolated and characterized the dbv clusters, involved in the formation of the glycopeptides A40926. The development of a gene-transfer system for Nonomuraea sp. has allowed the manipulation of the A40926 pathway. New derivatives were obtained by inactivating selected dbv genes. In addition, our data suggest differences in the biosynthetic routes for heptapeptide formation between the vancomycin and the teicoplanin families of glycopeptides.     

Isolation and characterization of bovine haptoglobin from acute phase sera

A macromolecular hemoglobin-binding protein, which was not detectable in normal bovine sera but appeared during acute phase inflammation, was purified, characterized, and designated as bovine haptoglobin (Hp). The purified protein had a molecular mass of 1,000-2,000 kDa, and was composed of two kinds of peptides, a 20-kDa peptide (alpha chain) and a 35-kDa glycopeptide (beta chain) linked by disulfide bonds. Amino acid composition and N-terminal sequence analyses revealed that both peptides were homologous to each counterpart of human Hp. Studies using some reducing reagents proved that highly polymerized Hp in serum was composed of 2-20 polymerized forms of alpha 2 beta 2 tetramer. Hp could bind one molecule of hemoglobin/alpha 2 beta 2 unit. Hp with smaller sizes obtained from native Hp by partial reduction with cysteine showed almost the same Hb-binding capacity.     

Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human beta-hexosaminidase

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.     

Asparagine-linked oligosaccharides in murine tumor cells: comparison of a WGA-resistant (WGAr) nonmetastatic mutant and a related WGA-sensitive (WGAs) metastatic line

MDW40, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic tumor cell line called MDAY-D2, is restricted to local growth at the subcutaneous site of inoculation. The WGAr tumor cells acquire metastatic ability by fusing spontaneously with a normal host cell followed by chromosome segregation, a process accompanied by reversion of the WGAr phenotype (i.e., WGAs). Since lectin-resistant mutant cell lines often have oligosaccharide alterations that may affect membrane function and consequently metastatic capacity, we compared the major Asn-linked glycopeptides in WGAr and WGAs cell lines. [2-3H]mannose-labeled glycopeptides were separated into four fractions on a DEAE-cellulose column and then further fractionated on a concanavalin A-Sepharose column. Glycopeptide structures were determined by: (a) sequential exoglycosidase digestion followed by chromatography on lectin/agarose and Bio-Gel P-4 columns and (b) proton nuclear magnetic resonance analysis. The metastatic WGAs cells had a sialylated poly-N-acetyllactosamine-containing glycopeptide which was absent in the nonmetastatic mutant cell line. Unique to the mutant was a neutral triantennary class of glycopeptide lacking sialic acid and galactose; the WGAr lesion therefore appeared to be a premature truncation of the antennae of the poly-N-acetyllactosamine-containing glycopeptide found in the WGAs cells. High mannose glycopeptides containing five to nine mannose residues constituted a major class in both WGAr and WGAs cells. Lysates of both wild-type and mutant cells had similar levels of galactosyltransferase activity capable of adding galactose to the N-acetylglucosamine-terminated glycopeptide isolated from mutant cells; the basis of the WGAr lesion remains to be determined.     

Glycopeptide dendrimers. Part I.

Glycopeptide dendrimers are branched structures containing both carbohydrates and peptides. Various classes of these compounds differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. The main stress is given to glycopeptide dendrimers, namely multiple antigen glycopeptides (MAGs). In MAGs, the core, branches or both are composed of amino acids or peptides. Other classes of glycodendrimers (PAMAM, polypropylene imine, cyclodextrin, calixarene, etc.) are mentioned too, but to a smaller extent. Their syntheses, physicochemical properties and biological activities are given with many examples. Glycopeptide dendrimers can be used as inhibitors of cell surface protein-carbohydrate interactions, intervention with bacterial adhesion, for studying of recognition processes, diagnostics, imaging and contrast agents, mimetics, for complexation of different cationts, as site-specific molecular delivery systems, for therapeutic purposes, as immunodiagnostics and in drug design. Biomedical applications of glycopeptide dendrimers as drug and gene delivery systems are also given.     

A simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano LC coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry

In this report we describe an on-column method for glycopeptide enrichment with cellulose as a solid-phase extraction material. The method was developed using tryptic digests of several standard glycoproteins and validated with more complex standard protein digest mixtures. Glycopeptides of different masses containing neutral and acidic glycoforms of both N- and O-linked sugars were obtained in good yield by this method. Upon isolation, glycopeptides may be subjected to further glycoproteomic and glycomic workflows for the purpose of identifying glycoproteins present in the sample and characterizing their glycosylation sites, as well as their global and site-specific glycosylation profiles at the glycopeptide level. Detailed structural analysis of glycoforms may then be performed at the glycan level upon chemical or enzymatic release of the oligosaccharides. Aiming at complementing other purification methods, this technique is extremely simple, cost-effective, and efficient. Glycopeptide enrichment was verified and validated by nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining electron-transfer dissociation (ETD) and collision-activated dissociation (CAD) fragmentation techniques.     

Structure of the high mannose oligosaccharides of a human IgM myeloma protein. I. The major oligosaccharides of the two high mannose glycopeptides.

The structures of the predominant high mannose oligosaccharides present in a human IgM myeloma protein (Patient Wa) have been determined. The IgM glycopeptides, produced by pronase digestion, were fractionated on DEAE-cellulonalysis shows that glycopeptide I contains Asn, Pro, Ala, Thr, and His and glycopeptide II contains Asn, Val, and Ser, which are the same amino acids found in the sequences around Asn 402 and Asn 563 respectively, to which high mannose oligosaccharides are attached in IgM (Patient Ou) (Putnman, F.W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (1973) Science 182, 287-290). The high mannose glycopeptides in IgM (Wa) exhibit heterogeneity in the oligosaccharide portion. Structural analysis of the major oligosaccharides indicates that the simplest structure is: (see article of journal). The larger oligosaccharides present have additional mannose residues linked alpha 1 yields 2 to terminal mannose residues in the above structure. Glycopeptide I contains primarily Man5 and Man6 species, while glycopeptide II contains Man6 and Man8 species. The two Man6 oligosaccharides have different branching patterns.     

Rat serum factors inhibiting the G1-S transition in hepatocytes. II. Properties of the low molecular weight factor

The properties were investigated of low molecular weight factors acting on the G1-S transition of baby rat hepatocytes. These factors were produced by incubating adult rat serum with trypsin or a 100,000 g liver microsomal fraction, and isolated by ultrafiltration. Enzyme degradation assays indicated that the active compound was in both cases a glycopeptide sensitive to pronase and papain and to the combination of neuraminidase and beta galactosidase. No loss of hepatocyte G1-S inhibitory activity was observed after heat treatment at pH 7.0. G50 gel filtration showed that both the G1-S inhibitory factors were collected in the last fractions eluted. The elution volume was slightly larger for the trypsin than for the microsomal-induced factor, suggesting a small difference between their molecular weight. These factors are believed to be glycopeptides with a molecular weight around 1400. They might be composed of a 3-sugar polysaccharide chain with a galactose preterminal and a neuraminic acid terminal, linked to a polypeptide chain of 6 to 8 amino acids.     

Evidence for a site-specific fucosylation of N-linked oligosaccharide of immunoglobulin A1 from normal human serum

Glycopeptides containing the N-linked oligosaccharide from human serum IgA1 were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). Two glycopeptides, GP1 and GP2, prepared from the endoproteinase Asp-N digest of the IgA1 heavy chain, were derived from the CH2 domain (N-glycan site at Asn263) and the tailpiece portion (N-glycan site at Asn459), respectively. The structure of the attached sugar chain was deduced from the mass number of the glycopeptide and confirmed by a two-dimensional mapping technique for a pyridylaminated oligosaccharide. GP1 was composed of two major components having a fully galactosylated bianntena sugar chain with or without a bisecting N-acetylglucosamine (GlcNAc) residue. On the other hand, the GP2 fraction corresponded to the glycopeptides having a fully galactosylated and fucosylated bianntena sugar chain partly bearing a bisecting GlcNAc residue. Thus, the site-specific fucosylation of the N-linked oligosaccharide on the tailpiece of the 1 chain became evident for normal human serum IgA1.