A. Gottschalk

Studies of mucoproteins I. The structure of the prosthetic group of ovine submaxillary gland mucoprotein

Abstract Ovine submaxillary gland mucoprotein (OSM), the most potent of the influenza virus haemagglutinin inhibitors, was found to contain equimolecular amounts of N-acetylneuraminic acid and N-acetylgalactosamine comprising 42% of the mucoprotein. The prosthetic group was isolated and shown to be the disaccharide 6-α- D -N-acetylneuraminyl-N-acetylgalactosamine by elementary analysis, identification of its components, susceptibility to α-neuraminidase and periodate consumption. The derivative 6-α- D -N-acetylneuraminyl-anhydro-N-acetyl-galactosamine, a chromogen in the direct Ehlrich reaction in the cold, formed from the prosthetic group on gentle alkali treatment, was also prepared. 93% of the total galactosamine of OSM was oxidized by periodate, and 85% of the N-acetylneuraminic acid was released by α-neuraminidase concomitant with the loss of biological activity.

Studies on mucoproteins III. The accessibility to trypsin of the susceptible bonds in ovine submaxillary gland mucoprotein

Ovine submaxillary gland mucoprotein was found to be susceptible to trypsin. The evidence is a marked increase in terminal NH2-groups and a corresponding rise in filterability through cellulose acetate membranes and an increase in electrophoretic mobility after trypsin treatment. Enzymic removal of the terminal neuraminic acid units of the prosthetic groups (6-α-D-N-acetylneuraminyl (2→6) N-acetylgalactosamine) increased the trypsin effect on ovine submaxillary gland mucoprotein by about 45%. This means that at least one-third of the trypsin-susceptible bonds in ovine submaxillary gland mucoprotein are inaccessible to the enzyme molecule. This figure may represent only a minimum value since any steric hindrance exerted by the remaining N-acetylgalactosamine unit cannot be assessed at present. n nThe complete loss of virus haemagglutinin inhibitory capacity of ovine submaxillary gland mucoprotein on trypsin action is interpreted in terms of a critical molecular size below which a mucoprotein ceases to act as an inhibitor.

Studies on mucoproteins VII. The linkage of the prosthetic group to aspartic and glutamic acid residues in bovine submaxillary gland mucoprotein

Abstract Treatment of bovine submaxillary mucoprotein with LiBH4 in tetrahydrofuran resulted in the reduction of approx. 83% of the total dicarboxylic acid residues and in the release of unreduced prosthetic groups previously identified as α- d -sialyl(2→6) N-acetylgalactosamine. When the acid hydrolysate of LiBH4-treated bovine submaxillary mucoprotein was fractionated according to Stein and Moore , fractions containing homoserine and α,δ-aminohydroxy-n-valeric acid were abtained. α,δ-aminohydroxy-n-valeric acid was identified by conversion to proline in acid medium; homoserine was identified by comparing its paper-chromatographic behaviour with that of authentic homoserine and by its resistance to periodate oxidation. From these data it was concluded that approx. 81% of the prosthetic groups in bovine submaxillary mucoprotein are joined through a glycosidic-ester linkage to the β-carboxyl group of aspartyl and the γ-carboxyl group of glutamyl residues respectively. The near completion of the reductive cleavage of these ester linkages was also indicated by the release of 84% of the prosthetic groups on alkali treatment of bovine submaxillary mucoprotein. The residual prosthetic groups are assumed to be linked by an O-glycosidic bond to serine and/or threonine. Homoserine and α,δ-aminohydroxy-n-valeric acid were synthesized by LiBH4 treatment of β-methyl aspartate and γ-methyl glutamate respectively.

V. The significance of n-acetylneuraminic acid for the viscosity of ovine submaxillary gland mucoprotein

Abstract Ovine submaxillary gland mucoprotein forms highly viscous solutions. The changes in viscosity with pH, time, concentration and with enzymic removal of the terminal neuraminic acid residues of the prosthetic group were investigated. After removal of N-acetylneuraminic acid by crystalline neuraminidase the viscosity of ovine submaxillary gland mucoprotein greatly decreased. A sharp drop in viscosity was also found immediately after lowering the pH in the range 7.8 to 1.7. Three-quarters of this drop in viscosity, shown to be reversible, was obtained by decreasing the pH from 4.4 to 1.7. These observations were interpreted as a folding of the extended molecule into a more compact configuration resulting from the removal or deionization of the strongly charged carboxyl groups of N-acetylneuraminic acid (p K a = 2.6). Independent of this sharp drop a slow decrease in viscosity due to aggregation was noted at all pH values. The bearing of these and previous findings on the possible configuration of the ovine submaxillary gland mucoprotein molecule is discussed.

IV. The linkage of the prosthetic group to the protein core in ovine submaxillary gland mucoprotein

Abstract When an unbuffered solution of ovine submaxillary gland mucoprotein, adjusted to pH 10.4, was heated for several hours at 80°, concomitant with the release of prosthetic groups ( α- d - N-acetylneuraminyl (2 → 6) N-acetylgalactosamine ) carboxyl groups at the residual OSM were unmasked. Since this observation suggested an ester type of linkage between the prosthetic group and the polypeptide chain, ovine submaxillary gland mucoprotein was submitted to treatment with LiBH4 in tetrahydrofuran, a reagent known to effect a reductive cleavage of ester linkages. To increase the solubility of ovine submaxillary gland mucoprotein in tetrahydrofuran, the size of the ovine submaxillary gland mucoprotein molecule was decreased by trypsin action and its dipolar ion character reduced by converting the free NH2-groups into their phenylthiocarbamyl derivatives. After treatment with LiBH4 of the ovine submaxillary gland mucoprotein frafments thus prepared, only 12% of the total dicarboxylic acids were recovered, indicating that about 80% of the prosthetic groups are involved in a glycosidic-ester linkage to the free carboxyl groups of aspartyl and glutamyl residues. The prosthetic group released on reductive cleavage of the ester bond was isolated. It was further shown that about 18% of the total N-acetylneuraminic acid remained bound within ovine submaxillary gland mucoprotein even after 4 h heating in 0.01 N NaOH, suggesting an alkali-stable O-glycosidic linkage of about 18% of the prosthetic groups to serine and/or threonine residues.

Studies on mucoproteins VIII. On the molecular size and shape of ovine submaxillary gland mucoprotein

A study is made of the particle dimensions and homogeneity of ovine submaxillary gland mucoprotein by viscosity, sedimentation velocity and Archibald measurements. The substance exhibits hypersharp sedimentation velocity patterns and is polydisperse; s°20,ω is 8.5 ± 0.8 S and [η] is 3.4 ± 0.1 dl/g; ν is 0.685 ± 0.01 ml/g. Treatment of the data by several methods indicates an average molecular weight for the preparations studied of (1.0 ± 0.2) · 106. The dimensions J, the ellipticity, and V′, the effective hydrodynamic volume, of the equivalent ellipsoid are calculated by the method of Ogston to be 8.7 and 30 ml/g, respectively. It is concluded that the mucoprotein is probably a random coil with some rigidity. X-ray data seem to support this conclusion. Problems in the application of the Archibald method to molecules like ovine submaxillary gland mucoprotein are discussed.

Studies of mucoproteins IX. On the susceptibility to alkali and to hydroxylamine of the predominant carbohydrate-peptide linkage in ovine-submaxillary-gland glycoprotein

Treatment of ovine-submaxillary-gland glycoprotein (OSM) with 0.1 N NaOH at 100° released about 89% of the carbohydrate groups (N-acetylneuraminyl (α,2 → 6)N-acetylgalactosamine; NANA ← GalNHAc) according to first-order reaction kinetics; k = 2.37·10−1 min−1 (half-time 2.9 min). The remaining prosthetic groups were liberated much more slowly; the first-order rate constant was 1.89·10−3 min−1. The carbohydrate-peptide linkages were also cleaved by NH2OH at pH 12.2 and 37° in a pseudo-monomolecular reaction, k = 2.3·10−3 min−1. The alkaline conditions on their own released the carbohydrate groups at a lower rate, k = 0.6·10−3 min−1. The disaccharides liberated were recovered quantitatively, mainly in form of their oxime which had protected them against destruction by alkali. Concomitant with the release of the disaccharides, hydroxamates were formed. Removal of the terminal negatively-charged N-acetylneuraminic acid (NANA) residues from OSM prior to NH2OH-treatment considerably increased the reaction rates: kNH2O− = 7·10−3 min−1, kOH− = 1.6·10−3 min−1. When about 89% of the carbohydrate groups had been liberated, the reaction was practically completed. OSM displayed no reducing power when tested with o-dinitrobenzene or Benedicts reagent under controlled conditions using GalNHAc as standard. n nThe data presented provide further proof for the previous conclusion that the bulk of the disaccharide groups in OSM are joined through a glycosidic-ester linkage to the peptide. 11% of the groups are apparently linked in another fashion; the possibility of an O-glycosidic linkage to serine residues for this fraction is discussed. The amide-N of OSM is low, 2.6 μmoles/100 mg OSM. It was shown that crystalline NANA, as was previously found for bound NANA, liberates NH3 under the acid conditions used in the amide determination of proteins.

Studies on mucroproteins: II. Analysis of the protein moiety of ovine submaxillary gland mucoprotein

Abstract Ovine submaxillary gland mucoprotein (OSM), shown previously to have the disaccharide α- d -N-acetylneuraminyl (2→6) N-acetylgalactosamine as prosthetic group, was analysed for its constituent amino acids and its amide content. OSM was found to have an almost complete complement of amino acids though with characteristic features. Its amide-N (0.34% of total N) could only be determined after enzymic removal of its terminal neuraminic acid residues since even on mild acid hydrolysis the release of amide-N was accompanied by the degradation of N-acetylneuraminic acid with liberation of about half its N as ammonia-N. OSM exhibited a single peak on electrophoretic examination and in the ultracentrifuge.

Studies on glycoproteins X. Equilibrium measurements on influenza virus-glycoproteins systems

Measurements in systems of enzymically inactive influenza viruses and sialoglycoproteins disclosed an equilibrium between the free reactants and the virus-glycoprotein complex. Equilibrium constants were determined by two methods: equilibrium filtration and ultracentrifugal separation. The glycoproteins tested were influenza virus haemagglutinin inhibitors of high potency (ovine submaxially glycoprotein) and low potency (fetuin, orosomucoid and trypsin-treated OSM). The equilibrium constants (in molar terms) were found to be 1·10−8 for OSM, 9·10−7 for fetuin, 2·10−5 for orosomucoid and 4·10−6 for the glycopeptides (assumed mol. wt. 50000) obtained by controlled trypsin treatment of OSM. Weight for weight OSM was 17 times more firmly bound to enzymically inactive Lee virus than were its constituent glycopeptides as formed on trypsin treatment. n nThe number of negatively charged terminal sialic acid residues attached simultaneously to complementary groupings at the virus surface, appears to determine primarily the inhibitory potency of the glycoproteins. This determinant is a compound of the number of sialic acid residues available per glycoprotein molecule and of the complementariness of the contacting areas on the surface of virus and glycoprotein.