Leopold März

Insect cells as hosts for the expression of recombinant glycoproteins

Baculovirus-mediated expression in insect cells has become well-established for the production of recombinant glycoproteins. Its frequent use arises from the relative ease and speed with which a heterologous protein can be expressed on the laboratory scale and the high chance of obtaining a biologically active protein. In addition to Spodoptera frugiperda Sf9 cells, which are probably the most widely used insect cell line, other mainly lepidopteran cell lines are exploited for protein expression. Recombinant baculovirus is the usual vector for the expression of foreign genes but stable transfection of - especially dipteran - insect cells presents an interesting alternative. Insect cells can be grown on serum free media which is an advantage in terms of costs as well as of biosafety. For large scale culture, conditions have been developed which meet the special requirements of insect cells.

Fucose in N-glycans: from plant to man

Fucosylated oligosaccharides occur throughout nature and many of them play a variety of roles in biology, especially in a number of recognition processes. As reviewed here, much of the recent emphasis in the study of the oligosaccharides in mammals has been on their potential medical importance, particularly in inflammation and cancer. Indeed, changes in fucosylation patterns due to different levels of expression of various fucosyltransferases can be used for diagnoses of some diseases and monitoring the success of therapies. In contrast, there are generally at present only limited data on fucosylation in non-mammalian organisms. Here, the state of current knowledge on the fucosylation abilities of plants, insects, snails, lower eukaryotes and prokaryotes will be summarised.

The asparagine-linked carbohydrate of honeybee venom hyaluronidase.

Hyaluronidase from the venom of the honeybee (Apis mellifera) has been purified by gelpermeation and cation exchange chromatography. Its asparagine-linked carbohydrate chains were released from tryptic glycopeptides with N-glycosidase A and reductively aminated with 2-aminopyridine. Separation of the fluorescent derivatives by size-fractionation and reversed-phase HPLC afforded eighteen fractions which were analysed by two-dimensional HPLC mapping combined with exoglycosidase digestions. The bulk of the N-linked glycans of hyaluronidase consisted of small oligosaccharides (Man1–3GlcNAc2), most of which were either α1,3-monofucosylated or α1,3-(α1,6-)difucosylated at the innermost GlcNAc residue. High-mannose type structures constituted the minor fractions, together making up about 5% of the oligosaccharide pool from hyaluronidase. Four fractions, making up 8% of the N-linked glycans, contained the terminal trisaccharide GalNAcβ1-4[Fucα1-3]GlcNAcβ1- in β1,2-linkage to the core α1,3-mannosyl residue. No evidence for the presence of O-glycans or sialic acids could be found.

Strict order of (Fuc to Asn-linked GlcNAc) fucosyltransferases forming core-difucosylated structures.

In insect cells fucose can be either α1,6- or α1,3-linked to the asparagine-bound GlcNAc residue of N-glycans. Difucosylated glycans have also been found. Kinetic studies and acceptor competition experiments demonstrate that two different enzymes are responsible for this α1,6- and α1,3-linkage of fucose. Using dansylated acceptor substrates a strict order of these enzymes can be established for the formation of difucosylated structures. First, the α1,6-fucosyltransferase catalyses the transfer of fucose into α1,6-linkage to the non-fucosylated acceptor and then the α1,3-fucosyltransferase completes the difucosylation.

PROCESSING OF ASPARAGINE-LINKED OLIGOSACCHARIDES IN INSECT CELLS : EVIDENCE FOR ALPHA -MANNOSIDASE II

The occurrence of α-d-mannosidase II activity in insect cells was studied using pyridylaminated oligosaccharides as substrates and two-dimensional HPLC and glycosidase digestion for the analysis of products. GlcNAcMan5GlcNAc2 was converted to GlcNAcMan3GlcNAc2 by each of the three cell lines investigated (Bm-N, Sf-21, and Mb-0503). The respective activity was highest in Bm-N cells which were used for further experiments. Man5GlcNAc2 was not degraded by the Bm-N cell homogenate. Thus, this α-mannosidase essentially exhibits the same substrate specificity as mammalian and plant Golgi α-mannosidase II. The α-mannosidase II-like activity from Bm-N cells exhibits a pH optimum of 6.0–6.5, has no requirement for divalent metal ions, and is highly sensitive to swainsonine. The α1,6-linked mannosyl residue is removed first as deduced from the elution time on reversed phase HPLC of the intermediate product. The same branch preference was found with α-mannosidase II from mung bean seedlings andXenopus liver. Upon ultracentrifugation of Bm-N cell homogenate, 72% of the mannosidase acting on the GlcNAcMan5GlcNAc2 substrate was found in the microsomal pellet indicating the enzyme to be membrane-bound

Evidence for the glycoprotein nature of the crystalline cell wall surface layer of Bacillus stearothermophilus strain NRS2004/3a

The surface layer of Bacillus stearothermophilus strain NRS2004/3a was isolated and chemically characterized. The results of these initial studies lead to the conclusion that the cell surface protein is glycosylated.

Two additional bacteriophage-associated glycan hydrolases cleaving ketosidic bonds of 3-deoxy-D-manno-octulosonic acid in capsular polysaccharides of Escherichia coli

Two bacteriophages degrading 3‐deoxy‐D‐manno‐2‐octulosonic acid‐(KDO)‐containing capsules of Escherichia coli strains were identified. Using modifications of the thiobarbituric acid assay, it was shown that each phage contains a glycan hydrolase activity cleaving one type of ketosidic linkage of KDO. Thus, the enzyme from phage ψ95 catalyses the hydrolysis of β‐octulofuranosidonic linkages of the K95 glycan; and ψ1092, the α‐octulopyranosidonic linkages of the K? antigen of E. coli LP1092. No cross‐reactivity of the phage enzymes with other KDO‐containing capsular polysaccharides was observed.

S12.20 Fucose in αl-3 linkage to the N-glycan core forms an allergenic epitope that occurs in plant and in insect glycoproteins

An important allergen was extracted from the mould Aspergillus fumigatus and purified by anion and cation exchange chromatography and gel filtration. The allergen, when examined by SDS-PAGE/immunoblotting, was found to be essentially homogeneous with a molecular weight of ca. 20.000 daltons. The allergen contained 3% protein and 90% carbohydrate. The carbohydrate moiety was composed of mannose, galactose and glucose in the ratio 10:7:0,9. The carbohydrate-protein linkage of the allergen is currently studied. Furthermore, we study how modifications of the molecule affect its allergenic activity.

S1.16N-glycan patterns and GlcNAc — Transferase I activities of three insect cell lines

bovine CD36 prepared from milk fat globule membranes (MFGM) derived from mammary gland epithelial cell membranes contains hybrid-type and hi-, triand tetraantennary complex-type sugar chains with the +_Neu5Aca2o6GalNAcfll-~4GlcNAc groups which amounted to 28% of the total sugar chains (1). Lectin blot analysis of bovine MFGM glycoproteins with use of Wistariafloribunda agglutinin (WFA) indicated that many glycoproteins including CD36 have the sugar chains with the groups. Structural analysis of the sugar chains released by hydrazinolysis from these glycoproteins by sequential exoglycosidase digestion and by methylation analysis revealed that the sugar chains which bound to a WFA-agarose column are mainly of biantennary complex-type and hybrid-type with the +Neu5Aca2~6 GalNAc/31--,4GlcNAc groups. In contrast, lectin blotting of glycoproteins obtained from primary cultured ceils derived from bovine mammary gland epithelial cells revealed that there were few glycoprotein bands stained with WFA. However, the bands appeared in the glycoprotein samples obtained from the cultured cells treated with prolactin, insulin and hydrocortison, suggesting that N-acetylgalactosaminylation of glycoproteins in bovine mammary gland epithelial cells is expressed by differentiation of cells under the influence of the hormones. (1) Nakata, N., Furukawa, K., Greenwalt, D. E., Sato, T. and Kobata, A. (1993) Biochemistry, in press.