No abstract

Two forms of protein-membrane anchor have been described for the externally disposed glycoproteins of eukaryotic plasma membranes; namely, the hydrophobic transmembrane polypeptide and the complex glycosylphosphatidylinositol (G-PI) moiety. The chemical structures of the major species of G-PI anchors found on a single variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei were determined by a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, chemical modification, and exoglycosidase digestions. The G-PI anchor was found to be heterogeneous with respect to monosaccharide sequence, and several novel glycosidic linkages were present. The results are pertinent to the mechanism of the biosynthesis of G-PI anchors.

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (.17 % total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11 % of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means.

Glycosyl-phosphatidylinositol (GPI) anchors have recently been identified as alternatives to hydrophobic amino acid sequences for the attachment of a variety of eukaryotic cell surface molecules to the lipid bilayer. In single cell eukaryotes the GPI group appears to be the predominant form of membrane attachment, and in vertebrates a substantial minority of molecules have this anchor including cell surface hydrolytic enzymes, antigens and cell adhesion molecules. Analysis of different GPI anchors suggests they share common structural features including linkage to the COOH group of the terminal amino acid via ethanolamine phosphate, the presence of phosphatidylinositol lipid and a glycan between the bridging ethanolamine phosphate and the lipid. In the case of the Trypanosoma brucie variant surface glycoprotein (VSG) the full structure of the GPI anchor has been determined and this provides a prototype for comparison with other molecules. We now report the structure of the GPI anchor of rat brain Thy-1 glycoprotein. It has an identical backbone to the VSG anchor but shows significant differences in side chain moieties.

Immunological competence is known to vary with age, and such age-related variations are often parabolic in nature . For example, both the level and avidity of the antibody response increase during postnatal development, reach a maximum in adulthood, and then decline during senescence (1). Cellular changes in the T lymphocyte compartment (both helper and suppressor T cell levels) appear to account for most variations in immunological competence (1, 2). Relatively few studies have attempted to correlate these functional changes with variations in the levels and nature of molecules expressed by immunocompetent lymphocytes. A study has therefore been undertaken to evaluate the glycosylation status of IgG during the natural history of human life .Human serum IgG has been reported to carry at least 30 different N-linked oligosaccharides, all of which are of the biantennary complex type, and most of which (>95%) carry N-acetylglucosamine in both outer arms (3, 4). Structural differences between these arise primarily from core-substitutions (i .e., fucose and the`bisecting' G1cNAc), outer-arm galactosylation, and sialylation (Fig. 1 a). We have previously reported that the percentage incidence of agalactosyl structures on the serum IgG of a group of patients with active rheumatoid arthritis (mean age 62 yr) was -51%, placing this group well above the average for a group of normal individuals (3, 5) .We now report that the galactosylation of IgG N-linked oligosaccharide changes as a parabolic function of age. This seemingly parallels changes in immunological competence and draws attention to the need to monitor agedependent variations in the expression of oligosaccharides in addition to those related to differences in cell type (6). Volume 167 May 1988 1731-1736 Materials and Methods Brief Definitive ReportSerum was obtained from 151 individuals of both sexes and varying in age from 1-70 yr . None of the individuals >15 yr old were known to have any pathological abnormalities, nor to have had any history of autoimmune disease, although some of the older individuals may have had subclinical levels of age-related diseases (such as osteoarthritis), but none has as yet presented with major symptoms. All children under the age of 15 yr who were studied were undergoing ear, nose, and throat surgery, hernia repairs, or treat-R. B. Parekh, R. A. Dwek, and T. W. Rademacher are members of the Oxford Glycobiology Unit, which is supported by the Monsanto Co .