Bernard Bayard

Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human serotransferrin

In previous papers [l-4] we have described 2 glycopeptides (Carbohydrate -+ Asn-Lys and Ser-Asn c Carbohydrate) isolated from pronase digests of human asialo-serotransferrin and we have demonstrated that in both glycopeptides the glycans were bound through a 4-N-(2-acetamido-2-deoxy-/3-D-glucopyranosyl)L-asparagine linkage. Evidence that the 2 glycans proceed from two different parts of the polypeptide chain was demonstrated by determination of amino-acid sequences of tryptic and chymotryptic glycopeptides : glycan I is conjugated in the N-terminal part of the peptidic chain and corresponds to the dipeptide Asn-Lys whereas glycan II is conjugated in the C-terminal part and corresponds to the dipeptide Ser-Asn [5-81. In this paper we report the primary structure of glycans I and II determined by application of different methods: exhaustive methylation, mild hydrolysis, acetolysis, hydrazinolysis as well as use of specific glycosidases.

Microheterogeneity of rat, mouse and human α1-fetoprotein as revealed by polyacrylamide gel electrophoresis and by crossed immuno-affino-electrophoresis with different lectins

Polyacrylamide gel electrophoresis and crossed immuno-affino-electrophoresis with several free lectins have been used to characterize and to compare the molecular heterogeneity of rat, mouse and human alpha1-fetoproteins. Each alpha1-fetoprotein contains a variable number of electrophoretic variants depending on the gel porosity. In SDS electrophoresis, two molecular size populations are present in rat alpha1-fetoprotein (Mr = 74 000 and 72 000) and in mouse alpha1-fetoprotein (Mr = 73 000 and 72 000) but only one is observed in human alpha1-fetoprotein (Mr = 70 000). The crossed immuno-affino-electrophoresis patterns square with affinity chromatography results and reveal a marked and characteristic heterogeneity for the three alpha1-fetoprotein species with Concanavalin A, Ricinus communis and Lens culinaris lectins. No lectin-alpha-fetoprotein interaction is apparent with Ulex, Lotus and wheat germ lectins. Since similar patterns are obtained whether with purified alpha1-fetoprotein or with unfractionated fresh fetal sera, it is likely that this heterogeneity is not a consequence of artefactual molecular modifications arising during the purification procedure.

Characterization and isolation of nine rat alpha-fetoprotein variants by gel electrophoresis and lectin affinity chromatography

Summary AFP purified from Rat amniotic fluid by immunosorbent chromatography, can be divided into nine different microheterogeneous forms. Preparative gel electrophoresis is first used for obtaining the two AFP A and AFP B populations. Upon affinity chromatography on RCA I -Sepharose, the slow electrophoresic form, AFP A , consists for 90 % of an unbound fraction (A 1 ) and 10 % of a bound fraction (A 2 ). Every one of the three AFP fractions (A 1 , A 2 and B) are then resolved on Con A-Sepharose in three new AFP components : Con A-non reactive (A 1a , A 2a and B a ) ; Con A-weakly reactive (A 1b , A 1b , B b ) ; Con A-reactive (A 1c , A 2c , B c ). The molecular nature of this heterogeneity is discussed.

Hydrazinolysis and nitrous acid deamination of the carbohydrate moiety of α1-acid glycoprotein

Hydrazinolysis followed by nitrous acid deamination of alpha1-acid glycoprotein gave acidic and neutral mono- and oligo-saccharides that contain 2,5-anhydro-D-mannose as reducing residue: alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-beta-D-Manp-(1 leads to 4)-2,5-anhydro D-mannose (1), beta D-Galp-(1 leads to 4)-2,5-anhydro-D-mannose (3), 2,5-anhydro-D-mannose, and two N-acetylneuraminic acid-containing oligosaccharides having the common partial sequence: NeuNAc-(2 leads to ?)-[BETA-D-Galp-(1 leads to 4)-2,5-anhydro-D-mannose] (5). This specific cleavage of 2-amino-s-deoxy-D-glucosyl linkages released almost quantitatively a very limited number of saccharides. Reduction with sodium borotritide of the products of cleavage allowed the precise determination of the molar proportion of 1, 3, and free 2,5-anhydro-D-mannose.

Evidence for uniformity of the carbohydrate chains in individual glycoprotein molecular variants.

Abstract Pooled and alkylated α 1 -acid glycoprotein was fractionated on a Con A-Sepharose column into two fractions : Con A-non reactive and Con A-reactive. The carbohydrate moiety from the α 1 -acid glycoprotein Con A-reactive variant, obtained by hydrazinolysis and quantitative re-N-acetylation, contains only identical two-branched oligosaccharide chains. From the present work on α 1 -acid glycoprotein and from previous studies on α 1 -fetoprotein, one can assume that glycoprotein glycosylation occurs uniformly along each polypeptide chain giving it identical oligosaccharide units at each glycosylation site.

Rat alpha-fetoprotein heterogeneity comparative chemical study of the two electrophoretic variants and their ricinus lectin-binding properties

Two electrophoretic forms of rat alpha-fetoprotein were purified using immunosorbent chromatography and preparative electrophoresis on polyacrylamide gel slabs. Some of their respective chemical properties and their affinity for the Ricinus communis lectin (RCAI) were compared. Electrophoresis on polyacrylamide gradient gel in the presence of sodium dodecyl sulfate indicated a slight difference in molecular 74 000 for the slow alpha-fetoprotein (AFPA) and 72000 for the fat alpha-fetoprotein (AFPB). no significant difference in amino acid composition between AFPA and AFPB was found. A residue of valine was identified at the C-germinal end of both alpha-fetoproteins. The analysis of the CNRr-cleavage products reveals slight differences between AFPS and AFPB. The slow moving alpha-fetoprotein could be further fractionated on RCAI-sepharose column in two components, AFPA1 and AFPA2 differing by their sialic acid content.

Electrophoretic preparation of the two rat α-fetoprotein variants

Alphafetoprotein (AFP) has already been purified from various sources by several convenient methods [l-4]. Rat AFP has been found heterogeneous on electrophoresis in low porosity polyacrylamide gels [1,4-61. However, the nature of the microheterogeneity remains unclear due to the difficulty to purify two AFP variants in sufficient amounts. Using a simple preparative procedure of polyacrylamide gel electrophoresis we were able to isolate the two Rat AFP and to compare some of their respective biochemical and immunological properties.

Glycan uniformity within molecular variants of transferrin with distinct affinity for concanavalin A

Abstract Human serum Transferrin has been fractionated on concanavalin A-Sepharose column into three fractions: Con A-non reactive (5 %), Con A-weakly reactive (30 %) and Con A-reactive (65 %). Each Con A-affinity transferrin variant contains a pair of identical oligosaccharide units either tri-branched in the Con A-non reactive variant or two-branched in the Con A-weakly reactive and Con A-reactive transferrin. This is an additional support to our proposal that glycosylation occurs uniformly along each polypeptide chain yielding identical oligosaccharide units at each glycosylated site.

Études sur les glycoprotéines. XLVIII. Description d'un procédé de fractionnement des acétolysats

Abstract Acetolysis, followed by quantitative de- O -acetylation with sodium methylate of the chloroform extract of the acetolyzates and chromatographic fractionation, was applied to the sialoglycopeptide α and asialoglycopeptide β obtained by pronase hydrolysis of ovomucoid. The acetolysis yielded small amounts of monosaccharides and a large proportion of oligosaccharides without transglycosylation. It does not split off the acetamido groups and, on the other hand, the sialosyl and the 2-acetamido- N -( L -aspart-4-oyl)-2-deoxy-β- D -glucopyranosylamine bonds are protected to a high degree. After de- O -acetylation, three fractions are obtained from the chloroform phase in the case of a sialoglycoprotide and two in the case of an asialoglycoprotide by chromatography on ion-exchange resins. The first fraction, not retained, contains neutral oligosaccharides from the median portion of the carbohydrate moieties. The second fraction, present only in the acetolyzates of the sialoglycopeptides and released from the anion-exchange resin, contains sialo-oligosaccharides from the outer part of the carbohydrate moieties. The last fraction, eluted from the cation-exchange resin, contains the glycopeptides and represents the carbohydrate components near the site of attachment to the peptide chain.

Rat alpha-fetoprotein heterogeneity: Affinity chromatography on Ricinus communis Sepharose column

The interaction between Ricinus communis agglutinin (RCA) with whole alpha-fetoprotein (AFP) and with its electrophoretic variants by affinity chromatography using Sepharose coupled lectin has been examined. AFP was isolated from amniotic fluid fetal newborn sera and hepatoma bearing rat serum and was purified by affinity chromatography on anti AFP-Sepharose column. The data demonstrate that RCA1-reactive AFPA2 had a lower content of sialic acid (4 residues/mole) as compared to the RCA1 nonreactive AFPA1 (6 residues/mole). It is postulated that the lack of 2 N-acetylneuraminic acid residues exposes 2 D-galactose terminal sites in the reactive form allowing it to react with the Ricinus lectin. This observation was confirmed by enzymic desialation of AFPA which unmasks all D-galactopyranosyl sites and allows the quantitative binding of the desialated variant. AFPB glycans appeared to be less accessible as shown by their incomplete in vitro enzymic desialation and their nonreactivity with the Ricinus lectin. AFP1 is invariably found in fresh sera or adult hepatoma sera and is not an artefact due to the method of isolation. Dosage of these proteins in the serum of rats from birth until Day 24 revealed that AFPB and AFPA2 disappear faster than the AFPA1 which may signify either that they become preferentially localized in certain organs or that they are more rapidly catabolized in the sera.