Direct evidence for the presence of beta-hydroxyphenylalanine and beta-hydroxytyrosine in cutinase from Fusarium solani pisi

Extracellular cutinase induced by cutin hydrolysate in glucose-grown Fusarium solani f. pisi was isolated in electrophoretically homogeneous form. This enzyme was similar to cutinase I generated by cutin-grown cells in its catalytic properties such as pH optimum, substrate specificity, and inactivation by “active serine”-directed reagents. Its molecular weight was 26,300 and this enzyme had a much larger content of serine and threonine residues than that found in cutinase from the cutin-grown cells. The hydrolysate-induced enzyme was a glycoprotein containing 6% carbohydrates. Alkaline NaB³H₄ treatment of the protein generated labeled protein and labeled carbohydrates. Analyses of the hydrolysates of these labeled products showed that alanine, α-aminobutyrate, phenylalanine, and tyrosine in the protein were labeled strongly suggesting that serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine were involved in O-glycosidic linkages in this protein. The protein hydrolysate also contained labeled gulonic acid, suggesting that d-glucuronic acid was attached to the protein via a base stable linkage, presumably an amide linkage at the N-terminus. The labeled reduced carbohydrates were identified by ion-exchange, thin-layer, gas-liquid, and high-performance liquid chromatographic techniques as mannitol, arabitol, gulonic acid, and 2-aminosorbitol. Thus mannose, arabinose, glucuronic acid, and glucosamine (possibly N-acetylated) were attached O-glycosidically to the hydroxyamino acids. Induction of cutinase by cutin hydrolysate in the presence of tritiated phenylalanine gave labeled cutinase. Cleavage of the O-glycosidically attached carbohydrates by anhydrous HF, followed by enzymatic hydrolysis of the labeled protein, gave rise to labeled amino acids, which upon analysis with an amino acid analyzer revealed four radioactive components. Two of them were identified as phenylalanine and tyrosine, while the other two cochromatographed with authentic β-hydroxyphenylalanine and β-hydroxytyrosine not only by the amino acid analyzer but also upon thin-layer chromatography. These results constitute the first direct evidence for the presence of the novel β-hydroxyaromatic amino acids in a protein.     

An effect of ATP on the permeability of transport-competent plasma membrane vesicles from mouse fibroblasts.

Methods for predicting peptide chain conformation have been applied to amino acid sequences adjacant to the carbohydrate attachment sites of glycoproteins containing the $\text{N}$-glycosylamine type of protein-carbohydrate linkage. Of 31 glycosylated residues examined 30 occur in sequences favouring turn or loop structures. Twentytwo of the glycosylated asparagine residues occur in tetrapeptides predicted to have the β-turn conformation. Carbohydrate attachment is therefore associated with peptide sequences which favour the formation of β-turn or other turn or loop structures.     

Coronavirus glycoprotein E1, a new type of viral glycoprotein

The carbohydrate contents of coronavirus glycoproteins E1 and E2 have been analyzed. E2 has complex and mannose-rich-type oligosaccharide side-chains, which are attached by N-glycosidic linkages to the polypeptide. Glycosylation of E2 is initiated at the co-translational level, and it is inhibited by tunicamycin, 2-deoxy-glucose, and 2-deoxy-2-fluoro-glucose. Thus, E2 belongs to a glycoprotein type found in many other enveloped viruses. E1, in contrast, represents a different class of glycoprotein. The following observations indicate that its carbohydrate side-chains have 0-glycosidic linkage. (1) The constituent sugars of E1 are N-acetylglucosamine, N-acetylgalactosamine, galactose, and neuraminic acid; mannose and fucose are absent. (2) The side-chains can be removed by β-elimination. (3) Glycosylation of E1 is not sensitive to the compounds interfering with N-glycosylation. E1 is the first viral glycoprotein analyzed that contains only 0-glycosidic linkages. Coronaviruses are therefore a suitable model system to study biosynthesis and processing of this type of glycoprotein.     

Methylation analysis of cell wall glycoproteins and glycopeptides from Chlamydomonas reinhardii

Cell wall glycoproteins from Chlamydomonas reinhardii and the glycopeptides produced by the action of thermolysin were subjected to standard methylation analysis. GC-MS of the methylated alditol acetates revealed short oligosaccharides some of which show branching. O-glycosidically linked galactofuranosyl residues are present. The asymmetric distribution of the major O-glycosidic linkages is also reported.     

Structural organization of the carbohydrate moiety of human transcortin as determined by methylation analysis of the whole glycoprotein

Methylation analysis of human transcortin showed that this glycoprotein contains N-glycosidically linked oligosaccharide chains of N-acetyllactosamine type, most of the chains being biantennary and others tri- and/or tetraantennary. The carbohydrate chains of transcortin are also heterogenous with respect to the content of fucose and the position of the glycosidic linkages.     

Purification and characterization of the major sialoglycoproteins of the rat erythrocyte membrane

The major sialoglycoproteins of the rat erythrocyte membrane were purified by hot phenol partitioning followed by cation-exchange chromatography on SP-Sephadex. Further purification was obtained by extraction with n-butanol and anion-exchange chromatography on DEAE-cellulose. The resulting sialoglycoprotein fraction was free of lipids and nonsialylated glycoproteins and gave rise to four major periodic acid-Schiff staining bands when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fastest migrating protein on these gels with an apparent molecular weight of 19,000 was purified to homogeneity by gel filtration. The amino acid and sugar compositions of these materials are reported. The protein moiety is rich in serine, threonine, and hydrophobic amino acids and the carbohydrate moiety is high in sialic acid and N-acetylgalactosamine. Most of the carbohydrate is linked O-glycosidically to serine and threonine residues, as shown by susceptibility to base-catalyzed β-elimination and concomitant reduction of serine and threonine to alanine and α-aminobutyric acid and of N-acetylgalactosamine to N-acetylgalactosaminitol in the presence of reducing agents. The significance of these data in light of the known role of the rat erythrocyte membrane sialoglycoproteins in erythropoiesis is discussed. The properties of the rat erythrocyte membrane sialoglycoproteins are compared to those of other species.     

Capillary gas chromatography of methylhexitol acetates obtalned upon methylation of N-glycosidically linked glycoprotein oligosaccharides

The 30 O-methylated hexitol and 2-deoxy-2-(N-methyl)acetamidohexitol acetates which commonly may be obtained during methylation analysis of N-glycosidically linked glycoprotein oligosaccharides or their biosynthetic precursors, were subjected to chromatography through glass capillary columns, wall-coated with either Silar 9CP, Dexsil 410, SE-30, or OV 101. All 22 methylhexitol acetates (e.g., 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and -mannitol) were optimally resolved on the (most polar) Silar column, whereas the 8 aminohexitol derivatives (as well as the corresponding unmethylated hexitol and aminohexitol acetates) were best separated on either Dexsil 410 or OV 101.     

Isolation of a collagen fraction from the body-wall glycoproteins of the leech (Hirudo medicinalis), and characterization of its carbohydrate-amino acid portion

Fractionation of the leech (Hirudo medicinalis) body-wall glycoproteins yielded a collagen fraction containing only d-glucose and d-galactose as its carbohydrate constituents. Digestion of the collagen with trypsin and pronase, and alkaline degradation of the resulting glycopeptides, gave a product that contained a disaccharide linked to hydroxylysine. Mild, acid hydrolysis of the N-acetylated glycopeptides yielded a disaccharide consisting of a d-glucose and a d-galactose residue. Various chemical and enzymic reactions of the disaccharide, the glycosyloxylysine, and the glycopeptide fraction indicated that the disaccharide is 2-O-α-d-glucopyranosyl-d-galactose, and that this is β-glycosidically linked to O-5 of the hydroxylysine residue in the collagen.     

Temporal aspects of the O-glycosylation of Saccharomyces cerevisiae mannoproteins

Cleavage of the O-glycosyl bonds of Saccharomyces cerevisiae cell wall mannoproteins by β-elimination resulted in the release of about 8% of the carbohydrate in the form of mannose and other low molecular weight oligomannosaccharides (mannose to mannopentaose), leaving 92% mannose still covalently linked to the peptide, and suggesting that this alkali-resistant fraction was N-glycosidically linked. At the non-permissive temperature, S. cerevisiae sec mutants accumulated in the cytoplasm mannoproteins with different degrees of O- and N-glycosylation. The glycoproteins of mutant sec 20-1 contained 60% of the carbohydrate linked by N-bonds, the remainder being O-glycosidically linked. Strains sec 19-1 and sec 1-1 contained 80 and 87%, respectively, of the mannose as N-linked carbohydrates. In addition, when the non-permissive conditions were prolonged over 2 h, strain sec 20-1 completed the formation of the O-linked oligosaccharides, suggesting that it is the kinetics of the process that determines the final composition of the mannoproteins. Our results suggest that the glycosylation of yeast cell wall mannoproteins is probably initiated in the lumen of the endoplasmic reticulum where the O-linked oligosaccharides may be completed. Maturation of the N-linked oligosaccharides, on the other hand, probably occurs during transport of the nascent mannoproteins to the cell surface.     

Purification and partial characterization of a procaryotic glycoprotein from the plasma membrane of Thermoplasma acidophilum

The obligate, thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum, grows optimally at 56° C and pH 2.0. Its plasma membrane possessed 21–22 protein bands that were resolved by polyacrylamide gel electrophoresis. One major membrane protein, molecular weight 152 000, which stained for carbohydrate with periodic acid-Schiff reagent, accounted for 32% (w/w) of the total membrane proteins. It was isolated and further purified by concanavalin A affinity chromatography. The carbohydrate content amounted to less than 10% (w/w) compared to that of the entire glycoprotein. The carbohydrate moiety consisted mainly of mannose residues with branched α 1 → 2 linkages at the non-reducing ends of the glycopeptide as determined by permethylation followed by gas chromatography-mass spectrometry analysis. The reducing end was an N-glycosidic linkage between asparagine and N-acetylglucosamine. The amino acid composition of this glycoprotein showed 62 mol% hydrophobic residues, while the acidic amino acid content contributed 9 mol% more than that of the basic amino acids. The existence of membrane glycoproteins in the procaryotic, wall-less T. acidophilum may provide a protective coat for the plasma membrane. The stereochemistry and the conformation of the carbohydrate chains, in conjunction with water turgor, may contribute to the rigidity of the membrane and the cation binding.     

Synthesis of guanidino sugar conjugates as GlcβArg analogs

The β-glucosyl linkage to the guanidine group of arginine (Arg) is found in amylogenin, a glycoprotein from sweet corn. Such a linkage is formed by a rare N-glycosylation of proteins. Synthesis of analogs of the unusual N-glycosidic linkage (GlcβArg) with an acetamido or triazole spacer between the glycosyl residue and the guanidine moiety was accomplished by the reaction of fully acetylated sugar unit containing a free amino group with bis-Boc-thiourea. Synthesis of N-glucosylarginine with an amido linker was also achieved during the present study. This methodology was also extended to the synthesis of cationic glucolipid.     

Quantitation of oligosaccharides released by the β-elimination reaction

A quantitative micromethod has been described for monitoring the rate and extent of the β-elimination reaction as applied to O-glycosyl-glycoproteins utilizing alkaline tritiated borohydride. The procedure simultaneously labels the released oligosaccharides by their reduction to the corresponding tritiated alditols. The alkaline tritiated borohydride treatment also results in the labeling of the protein moiety of the glycoprotein and this can be quantitatively separated from the carbohydrate moiety on a cation exchange resin; the carbohydrate moiety is not adsorbed, while the protein moiety is adsorbed and then eluted with HCl. The radioactivity in the aqueous eluate of the resin is therefore a direct measure of the amount of oligosaccharides released by the β-elimination reaction. The sensitivity of the method is dependent on the specific activity of the tritiated sodium borohydride used. The stoichiometry of the reaction has been established by the use of N-acetylgalactosaminyl-O-glycoproteins, demonstrating that at the completion of the β-elimination reaction: (a) none of the radioactivity attributable to the protein moiety contaminates the carbohydrate moiety, (b) all the carbohydrate components of the glycoprotein are found in the aqueous eluate from the cationic exchange resin, (c) all the radioactivity in this aqueous eluate is associated with the sugar known to be at the reducing end of the oligosaccharide chain bound to serine or threonine of the glycoprotein (in the examples discussed, N-acetylgalactosamine), and (d) there is no additional hydrolysis of the oligosaccharide chains during the processing.     

A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN^Lec1. We found that Sf-RVN and Sf-RVN^Lec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN^Lec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN^Lec1 cells were Endo H-cleavable Man₅GlcNAc₂ structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.     

A new method for degradation of the protein part of glycoproteins: isolation of the carbohydrate chains of asialofetuin

A new method has been developed for degrading the protein part of several glycoproteins, whilst leaving the carbohydrate portion virtually intact apart from partial degradation at the reducing end. The method is based upon stabilization of the glycosidic linkages of the sugar residues by trifluoroacetyl groups and subsequent cleavage of the peptide bonds by transamidation. The two reactions are carried out in a mixture of trifluoroacetic anhydride and trifluoroacetic acid. After O- and N-detrifluoroacetylation, the carbohydrate portion can be isolated and re-N-acetylated. The applicability of the method is demonstrated by the isolation from asialofetuin of the carbohydrate chains that are attached by N- and O-glycosyl links.     

Effect of distal sugars and interglycosidic linkage on the N-glycoprotein linkage region conformation: synthesis and X-Ray crystallographic investigation of β-1-N-alkanamide derivatives of cellobiose and maltose as disaccharide analogs of the conserved chitobiosylasparagine linkage

The linkage region constituents, 2-deoxy-2-acetamido-β-D-glucopyranose (GlcNAc) and L-asparagine (Asn) are conserved in the N-glycoproteins of all eukaryotes. Elucidation of the structure and conformation of the linkage region of glycoproteins is important to understand the presentation and dynamics of the carbohydrate chain at the protein/cell surface. Earlier crystallographic studies using monosaccharide models and analogs of N-glycoprotein linkage region have shown that the N-glycosidic torsion, ?_N, is more influenced by the structural variation in the sugar part than that of the aglycon moiety. To access the influence of distal sugar as well as interglycosidic linkage (α or β) on the N-glycosidic torsion angles, cellobiosyl and maltosyl alkanamides have been synthesized and structural features of seven of these analogs have been characterized by X-ray crystallography. Comparative analysis of the seven disaccharide analogs with the reported monosaccharide analogs showed that the ?_N value of cellobiosyl analogs deviate ~9° with respect to GlcβNHAc. In the case of maltosyl analogs, deviation is more than 18°. These deviations indicate that the N-glycosidic torsion is influenced by addition of distal sugar as well as with respect to inter glycosidic linkage (α or β); it is less influenced by changes occurring at the aglycon. The χ₂ value of alkanamide derived from glucose, cellobiose and maltose exhibit a large range of variations (from 1.6° to ?109.9°). This large span of χ₂ value suggests the greater degree of rotational freedom around C1′-C2′ bond which is restricted in GlcNAc alkanamides. The present finding explicitly proved the importance of molecular architecture in the N-glycoproteins linkage region to maintain the linearity, planarity and rigidity. These factors are necessary for N-glycan to serve role in inter- as well as intramolecular carbohydrate-protein interactions.     

A new neuraminic acid derivative and three types of glycopeptides isolated from the Cuvierian tubules of the sea cucumber Holothuria forskali

The Cuvierian tubules of Holothuria forskali Della Chiaje, a sea cucumber found in the Adriatic Sea, were investigated with regard to their carbohydrate moieties. From a Pronase digest of these tubules three types of carbohydrate units were isolated and characterized. 1. A high-molecular-weight glycopeptide fraction was shown to contain sulphated polyfucose, galactosamine, a uronic acid and a previously unknown neuraminic acid derivative. The sulphate was shown by i.r. analysis to be present as an O-ester. The carbohydrate unit was linked O-glycosidically to threonine and serine residues in the polypeptide chain. The hitherto unknown neuraminic acid derivative (Hf-neuraminic acid) was resistant to enzymic cleavage by neuraminidase, even after mild alkaline hydrolysis for the removal of O-acyl residues. However, the glycosidic linkage of this compound to the other part of the carbohydrate moiety was readily cleaved by mild acid hydrolysis. Its chromatographic properties distinguished Hf-neuraminic acid from other known neuraminic acid derivatives (N-acetyl-, NO-diacetyl-, NOO-triacetyl- and N-glycollyl-neuraminic acid). Further, this acidic sugar was shown to possess neuraminic acid as its basic structure. Thus, an as yet unknown substituent lends the distinct properties to Hf-neuraminic acid. 2. The carbohydrate composition of a second glycopeptide fraction consisting of a derivative of neuraminic acid, galactose, mannose and glucosamine was similar to that of the well-known carbohydrate groups of the globular glycoproteins. 3. The third fraction contained two glycopeptides containing the disaccharide, glucosylgalactose, which was shown to be linked to the hydroxyl group of hydroxylysine residues of a collagen-like protein. Approximately half of these residues were glycosylated. In addition to these glycopeptides, a small amount of a third glycopeptide that carried only a galactosyl residue was detected. The amino acid sequence of the two major compounds were found to be Gly-Ala-Hyl*-Gly-Ser and Gly-Pro-Hyl*-Gly-Asp, where Hyl* represents a glycosylated amino acid residue.     

Water-soluble glycoproteins from Cannabis sativa (South Africa)

The water-soluble glycoproteins obtained from Cannabis leaves of plants grown from South African seeds have been further studied. Treatment of the glycoprotein fractions with NaOH in the presence of NaBH₄, resulted in a significant decrease in the serine content and a corresponding increase in alanine. The carbohydrate side chains released contained the sugar alcohol, galactitol. By treatment of the glycoprotein fractions with NaOH in the presence of Na₂SO₃, and subsequent acid hydrolysis, cysteic acid was formed. These data indicate that carbohydrate and protein are connected via serine-O-galactoside linkages. Further investigation of the structure of the carbohydrate part of the glycoproteins was carried out by methylation analysis, Smith-degradation and enzyme incubation. The present glycoprotein material of plants grown from South African seeds is similar to the material previously investigated, but in contrast to the latter, it is devoid of hexosamine.     

Conformational study of α1-acid glycoprotein

The secondary structure of the protein moiety of the α₁-acid glycoprotein (orosomucoid) was evaluated from its primary structure by using the Lim and Chou and Fasman predictions, and the corresponding dichroic spectrum was calculated. The experimental dichroic spectrum of the whole glycoprotein was compared with the summation of (i) the calculated dichroic spectrum due to the protein moiety and (ii) the experimental dichroic spectrum of the carbohydrate moiety. The results are in good agreement with the fact that the carbohydrate moities do not produce any pertubation of the protein conformation. In addition, we observed that four out of five glycan chains are linked to Asn residues which are situated either in a reverse β-turn or in regions where charged and polar residues are numerous, that is, on the outside of the protein.     

Analysis of histidine-containing dipeptides, polyamines, and related amino acids by high-performance liquid chromatography: application to guinea pig brain.

A procedure is described for coupling wheat germ agglutinin to cyanogen bromide-activated Sepharose to yield a lectin affinity column of high capacity. Covalent linkage of the lectin to the insoluble matrix is carried out in the presence of a mixture of β-1,4-linked N-acetylglucosamine oligosaccharides prepared from chitin. The lectin-affinity column specifically recognizes glycoproteins containing N-acetylglucosamine residues with the capacity of binding 0.6–1.0 mg of ovomucoid per milliliter of gel. The affinity column is stable (as determined by ovomucoid binding) and shows little loss in binding capacity or specificity after repeated usage. Important characteristics for the use of this column to purify glycoproteins are described.     

The isolation and partial characterization of a glycoprotein isolated from human gastric aspirates and from extracts of gastric mucosae

The isolation and partial characterization of a glycoprotein isolated from individual gastric aspirates and extracts of gastric mucosae solubilized with N-acetylcysteine is described.The isolated glycoproteins and the glycoproteins from proteolysed gastric aspirates showed virtually the same carbohydrate and amino acid composition. The results indicate that they consist of a protein core to which are attached carbohydrate side-chains composed of four sugars: N-acetylgalactosamine N-acetylglucosamine, galactose, fucose showing a ratio of 1 : 3 : 4 : 2. Superimposed on this basic structure were additional sugar residues, the blood-group determinants. The results also suggest that the carbohydrate side-chains are linked by an alkali-labile O-glycosidic linkage to the threonine and serine residues of the protein core, N-acetylgalactosamine forming the link.