The O-linked oligosaccharides of the jelly coat surrounding the eggs of Xenopus laevis were analysed by 1H-NMR spectroscopy. Among the 12 neutral oligosaccharide-alditols which have been characterized, three of them possess the following unusual structures: [sequence: see text] As previously observed for six other amphibian species, the carbohydrate chains of the jelly coat of Xenopus eggs display a high species specificity which could support a biological role during the fertilization processes.

In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for ␤-1,2-oligomannosides and was biosynthetically labeled by [ 3 H]mannose, [3 H]palmitic acid, and [ 32 P]phosphorus. PLM has also been shown to be released from the C. albicans cell wall and to bind to and stimulate macrophage cells. In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C. albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids. We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis. Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1,2-linked mannose units. NMR studies evidenced linear chains of ␤-1,2-oligomannose as the major PLM components. Mass spectrometry analysis revealed that these chains were present in phosphoinositolmannosides with degrees of polymerization varying from 8 to 18 sugar residues. The PLM appears as a new type of eukaryotic inositol-tagged glycolipid in relationship to both the absence of glucosamine and the organization of its glycan chains. This first structural evidence for the presence of ␤-1,2-oligomannosides in a glycoconjugate other than the C. albicans phosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.

Phosphocalmodulin (PCaM) was identified after analysis of calmodulin (CaM) preparations by two-dimensional gel electrophoresis by using a modified ampholyte system to resolve very acidic proteins. The analysis of CaM prepared by the conventional procedure based upon its heat resistance and acidity as well as the analysis of whole urea extracts from brain showed that PCaM was a major component in this tissue. PCaM was 1 pH unit more acidic than CaM, and its electrophoretic mobility, unlike CaM, was not changed by either calcium or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. In urea extracts of brain prepared in buffers containing phosphate and sodium fluoride, PCaM was as prominent as CaM; it was partially converted into CaM after elution from the gel and reelectrophoresis. Amino acid analysis of PCaM and CaM purified by two-dimensional gel electrophoresis showed the same composition for the two proteins, including their trimethyllysine content. Incorporation of 32P occurred exclusively into the acidic variant when brain slices were incubated with H332PO4; amino acid analysis showed that the phosphate was bound to serine residues. CaM was found also to be phosphorylated in vitro by a phosphorylase kinase preparation from skeletal muscle.

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The development of therapeutic glycoprotein production using the baculovirus expression system depends on the ability of insect cell lines to reproduce site specific mammalian-like N-glycans. A combination of 1H-NMR and mass spectrometry techniques (MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate the N-linked oligosaccharides microheterogeneity on three different N-glycosylation sites, Asn233, Asn476, and Asn545, of a baculovirus-expressed recombinant bovine lactoferrin produced in Mamestra brassicae. Two families of N-glycan structures have been found: first, oligomannosidic glycans (Man[9-5]GlcNAc2) and secondly, short truncated partially fucosylated glycans (Man(3-2)[Fuc(0-1)]GlcNAc2). These results indicate that Mamestra brassicae cell line is not able to synthesize complex N-glycans, even if an alpha1,6-linked fucose residue is frequently present on the asparagine-bound N-acetylglucosamine residue of short truncated structures. Nevertheless, we have shown that Mamestra brassicae ensures the same N-glycosylation pattern as found on natural bovine lactoferrin showing the same distribution between complex and high-mannose type glycans on the different glycosylation sites. Sites which are naturally occupied by high-mannose glycans (Asn233 and Asn545) are substituted essentially by the same type of N-glycans in the recombinant counterpart, and the site Asn476,which carries sialylated complex type chains in the natural glycoprotein, is substituted by short, truncated, partially fucosylated chains in Mamestra brassicae-expressed bovine lactoferrin. These various results lead us to the conclusion that bovine lactoferrin is an interesting model to determine the potential of glycosylation of the baculovirus/insect cell expression systems.

Two octasaccharides, two dodecasaccharides and a tridecasaccharide have been isolated from human milk by a combination of paper chromatography, high-performance liquid chromatography and high-performance anion-exchange chromatography. Their structural analysis investigated by 400-MHz 'H-and 13C-NMR spectroscopy and by mass spectrometry led to the following structures.The two octasaccharides have been previously characterized in human milk; the complete assignment of the 'H and 13C spectra is reported here. One of the dodecasaccharides is a tetrafucosyl derivative with a new core : typeI-@1-3)-typeII-@1-3)-typeII-@1-3)-Gal(@1-4)Glc, where type1 = Gal@l-3)GlcNAc and type11 = Gal@ -4)GlcNAc. These oligosaccharides display simultaneously Lewisb and Lewis" determinants or Lewis", Lewisb and Lewis" determinants. Abbreviations. HPAEC, high-performance anion-exchange chromatography; FAB-MS, fast-atom-bombardment mass spectrometry; COSY, two-dimensional correlated NMR spectroscopy.

The identified structures corresponded to N-acetyllactosaminic biantennary glycans and were ~-2,3-disialylated forms (80%) or a-2,3-monosialylated (20%) forms. Moreover, 70% of total glycans were ~-l,6-fucosylated at the GIcNAc residue linked to asparagine. In regard to its glycan moiety, the recombinant glycoprotein is close to native lactoferrins from milk or leucocytes but shows specific structural features which should be taken into account prior to in vivo and in vitro biological studies.

The heat-stable acid-soluble phosphoglycoprotcin componcnt PP3 was isolated from the bovine milk proteose peptone fraction by concanavalin A affinity chromatography. Glycopeptidcs werc released by pronase digestion of the milk component PP3 and were subsequcntly separated by high-pH anion-exchange chromatography on Carbopat: PA-1. The prirnary structures of the glycan and peptide rnoieties of eight N-glycopeptides have been cstablished by combining inethylation analysis, mass spectronictry, 400-MHz 'H-NMR spectroscopy, and peptide sequence analysis. All the analyzed fractions contained biantcnnary N-acetyllactosamine-type carbohydrate chains. sottie of them with a GalNAc(@1-4)GlcNAc or u NeuAc(cx2-6)GalNAc(~1-4)GlcNAc group. This particular sequence did or did not rcplace the Gal@l-4)GlcNAc group usually found in most N-linked glycans. Moreover, the sialylated Gal and GalNAc rcsidues were only found on the Man(n1-3) antenna.Keywords: bovine milk; proteose peptone; component PP3 ; glycoprolein ; glycan.Component PP3 (protcose peptone 3), also named lactophorin by Kanno (1989), is a phosphoglycoprotein with an apparent molecular mass of 28 kDa isolated from the proteose peptone fraction of bovine milk (Sflrensen and Petersen, 1993). This component exhibits a strong surface activity at the oillwater interface of an ernulsion that prevents some lipase added to the emulsion from adsorbing at the interface and therefore inhibits lipolytic activity (Girardet et al., 1993;Courthaudon et al., 1995). The peptide primary structure has been determined (S@rensen and Petersen, 1993) and contains 135 amino acid residues. Component PP3 is identified as the bovine homologue of the murine glycosylation-dependent cell adhesion molecule I (glycosylation-dependent CAM-1 ; accession number LO81 01, GenBank database, release 87.0), a mucin-like endothelial cell surface ligand for the leucocyte adhesion molecule, L-selectin (Lasky et al., 1992; Johnsen et al., 1995). A glycoprotein with apparent molecular mass of 18 kDa is associated with component PP3 and corresponds to the 54-135 fragment of component PP3 released by plasmin hydrolysis in milk.Two 0-glycosylation (Thrl6 and Thr86) and one N-glycosylation (Asn77) sites were identified in component PP3. Two of them (Asn77 and Thr86) located in the C-terminal region of the protein are also found in the 18-kDa glycoprotein. Component PP3 is present in bovinc milk whey but possesses one or several common epitopes with glycoproteins of the milk fat globule membrane (MFGM; Kester and Brunner, 1982: Nejjar et al., 1986; Kanno, 1989 In this study, we thereforc investigate the slructurc or Nglycans of component PP3 in order to cornpnre lhese N-glycans to the known N-linked carbohydrate chains of the MFGM glycoproteins. EXPERlMENTAL PKOCEDUKESAffinity chromatography on immobilized concanavalin A (CunA). Raw milk sample was produced by a single animal with the following phenotype: a,,-casein BB, @-casein A,A,, ic-casein AB, u-lactalbumin AA, and j$-lactoglohulin RB. Proteose peptone extracts ...