Svein Haavik

Glycosylation of immunoglobulin light chains associated with amyloidosis

AL amyloidosis is a fatal disease caused by deposition of immunoglobulin light chains in a fibrillar form (AL) in various organs. By searching the Kabat database of immunoglobulin sequences using the KabatMan software, we have shown that there is a preponderance of the consensus glycosylation sequon (AsnXxxSer/Thr) in the framework regions of amyloid light chains. We have characterised by computer graphics simulations, NMR spectroscopy and carbohydrate biochemistry the structure and conformation of the oligosaccharide from amyloid protein AL MS (λI) and from the amyloid associated Bence Jones protein of patient MH (K1). These proteins have glycosylation in the hypervariable complementarity–determining region versus framework region, respectively. Both contained a 2–6 sialylated core fucosylated biantennary chain mostly with bisecting GlcNAc. Together our results suggest that light chain glycosylation may be one of several modifications which may render the protein more prone to amyloid formation.

Raspberry leaf--should it be recommended to pregnant women?

This review evaluates the safety and efficacy of raspberry leaf (Rubus idaeus) in pregnancy. The electronic databases PubMed, ISI Web of Science, AMED, EMBASE, Natural Medicines Comprehensive Database and Cochrane Library were searched. Altogether 12 original publications with focus on safety or efficacy during pregnancy, pharmacology and in vitro tests explaining mode of action or constituents in Rubus idaeus were reviewed. Limited documentation exists and part of it is 50 years old or older. Only the latest animal study indicates an increased risk for the unborn child; however, all the studies are small and cannot rule out negative effects on pregnancy outcome. The efficacy of raspberry leaf is not convincingly documented. The use of raspberry leaf in pregnancy is a traditional herbal therapy and is recommended by some midwives. Due to the lack of evidence for safety and efficacy such recommendations are questionable. Suggestions for future work are given.

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

Abstract The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α- l -arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α- l -arabinofuranosidase did not affect the allergenic activity of the extract.

Human carcinoma-associated antigen (HCA), isolated from the endometrial carcinoma cell line KLE-1 and ascitic fluid of a patient with ovarian carcinoma; comparison with epiglycanin.

Human carcinoma-associated antigen (HCA), detected by the mouse monoclonal anti-epiglycanin antibody, AE-3, has been isolated from ascitic fluid taken from a patient with metastatic ovarian adenocarcinoma and from spent medium of the human endometrial carcinoma cell line KLE-1 and compared with epiglycanin. The ascitic fluid and the spent medium were concentrated by a Filtron Ultrasette 100 K Omega membrane and fractionated by gel filtration on Sepharose CL-2B. The active fractions which consisted mainly of glycoproteins having relative molecular weights in the range 1000-2000 kDa compared to dextrans, were further purified by affinity chromatography on a column of immobilized AE-3. The active fraction was subjected to SDS-PAGE and blotted onto a PVDF membrane. The amino acid composition of HCA isolated from the two sources, were related but not identical and both showed some differences from the amino acid composition of epiglycanin. They all have, however, compositions typical of mucin-type glycoproteins. The isoelectric point for HCA from both KLE-1 and ascitic fluid were determined to be at pH 1.8 and the buoyant densities were about 1.4 g/ml as determined by cesium trifluoroacetate gradient centrifugation.

Specificity studies of an antibody developed against a mucin-type glycoprotein

The specificity of a new anti-epiglycanin antibody (AE-3) which recognizes a mucin-type glycoprotein, the Human Carcinoma Antigen, found in the blood of patients with carcinomas, was studied. Information regarding the chemical nature of the antibody binding site was obtained by altering the structure of epiglycanin by chemical or enzymic means and testing the product in a competitive binding assay for inhibition of the binding of AE-3 to epiglycanin. The need for a high molecular weight antigen containing clustered T disaccharide, Galβ1-3GalNAc, was demonstrated. The specificity was further explored by inhibition studies with glycopeptides having one to three mono- to disaccharides. The results were interpreted using computer graphics molecular modeling which predicted the specific recognition of hydroxyl groups on oligosaccharides on adjacent amino acids. Thus T antigen O-linked glycopeptide tumour markers can be designed to be distinguished by antibodies by the amount of clustering of their oligosaccharides.

The carbohydrate structure of the amyloid immunoglobulin light chain protein EPS

The glycosylated amyloid light chain protein EPS was isolated from amyloid fibrils from the liver of a patient with Waldenstroms macroglobulinemia. A tryptic glycopeptide of EPS (200 nmol) was isolated and characterized by 1H-n. m. r. The glycopeptide was digested with N-glycanase and the released oligosaccharides analyzed by high performance anion exchange chromatography. The monosaccharide composition and the linkage of the different oligosaccharides were determined by methanolysis and methylation analysis. The NMR spectrum revealed a bisected biantennary oligosaccharide as the main structure. HPAEC-PAD demonstrated the presence of several other related glycans in the glycopeptide preparation.