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Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish(Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried alpha2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.

Protein expression in foetal brain with or without chromosome 21 trisomy (Down's syndrome) was analyzed by two-dimensional gel electrophoresis and mass spectrometry. Data generated by in-gel digestion and matrix-assisted laser desorption/ionization mass spectrometry allowed identification of 40 proteins. Most of these are common to syndrome and healthy subjects and represent different types of protein. However, a few proteins, identified as truncated structural proteins (tubulin, actin), were present in part of the trisomy samples but absent from the controls. This is interpreted to indicate increased proteolysis in the syndrome samples but could also reflect some altered expression or processing. Independent of the apparently increased proteolysis in the syndrome samples, and in spite of the use of total brain tissues, the results show that two-dimensional protein separation patterns are largely similar between the syndrome and control samples upon silver-staining, but that differences associated with structural components can be detected and identified.

Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated. Toxins were purified from methanol extracts of the B. licheniformis biomass using boar sperm cells as the toxicity indicator. The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI-TOF-MS. The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino-acid residues L-Gln, L-Leu, D-Leu, L-Val, L-Asp, D-Leu and L-Ile in that order. Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3-hydroxy fatty acids are 13-15 carbons in length. We showed that the toxins from food and food poisoning isolates of B. licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa. Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity.

Multifucosylated sialo-polylactosamines are known to be high affinity ligands for E-selectin. PSGL-1, the physiological ligand of P-selectin, is decorated in HL-60 cells by a sialylated and triply fucosylated polylactosamine that is believed to be of functional importance. Mimicking some of these saccharide structures, we have synthesized enzymatically a bivalent [sialyl diLex]-glycan, Neu5Acalpha2-3'Lexbeta1-3'Lexbeta1-3'(Neu5Acalpha2-3'Lexbeta1-3Lexbe ta1-6')LN [where Neu5Ac is N-acetylneuraminic acid, Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc and LN is the disaccharide Galbeta1-4GlcNAc]. Several structurally related, novel polylactosamine glycans were also constructed. The inhibitory effects of these glycans on two L-selectin-dependent, lymphocyte-to-endothelium adhesion processes of rats were analysed in ex-vivo Stamper-Woodruff binding assays. The IC50 value of the bivalent [sialyl diLex]-glycan at lymph node high endothelium was 50 nm, but at the capillaries of rejecting cardiac allografts it was only 5 nm. At both adhesion sites, the inhibition was completely dependent on the presence of fucose units on the sialylated LN units of the inhibitor saccharide. These data show that the bivalent [sialyl diLex]-glycan is a high affinity ligand for L-selectin, and may reduce extravasation of lymphocytes at sites of inflammation in vivo without severely endangering the normal recirculation of lymphocytes via lymph nodes.

Matrix metalloproteinase-1 (MMP-1) is a collagenolytic metalloproteinase capable of cleaving native triple-helical forms of several collagen subtypes, as well as a number of non-collagenous substrates. The role of MMP-1 in various diseases affecting the connective tissue is well characterized. MMP-1 is secreted as both glycosylated and unglycosylated species, and the two forms have been shown to be identical with respect to substrate specificity, specific activity and inhibitory profile. No function for the glycan moiety of the enzyme has been ascribed to date. In the present study, we report on the detailed characterization of MMP-1-derived oligosaccharides. Using strategies based on sequential exoglycosidase digestion combined with matrix-assisted laser desorption ionization-time of flight MS and electrospray tandem MS, we have characterized the N-glycan structures of MMP-1, derived from human dermal fibroblasts and from the HT-1080 fibrosarcoma cell line. MMP-1 derived from fibroblasts was found to carry mainly alpha 2,3-sialylated complex-type diantennary glycans. On the other hand, HT-1080 cells produce MMP-1 that has a heterogeneous glycosylation pattern, comprising diantennary glycans carrying Lewis X, LacdiNAc, sialylated LacdiNAc and GalNAc beta 1,4 (Fuc alpha 1,3)GlcNAc (LacdiNAc analogue of Lewis X) as terminal elements. We also show that, of the two potential glycosylation sites in the MMP-1 sequence, only Asn120 is used.

We have previously isolated and partially characterised the components of a highly purified interferon-alpha (IFN-alpha) preparation produced by Sendai-virus-induced human peripheral blood leukocytes. Nine IFN-alpha species were identified, and two of these were found to be glycosylated [Nyman, T. A., Tölö, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon-alpha subtypes produced by Sendai-virus-induced human peripheral blood leukocytes, Biochem. J. 329, 295-302]. Here, we isolated the N-linked oligosaccharides of IFN-alpha14c and the O-linked chains of IFN-alpha2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The IFN-alpha14c N-glycans were shown to exhibit core-fucosylated biantennary glycans, with about 10% carrying an additional alpha1,3-linked fucose unit at the antennae. The IFN-alpha2b was shown to carry about 50% core type-1 disialyltetrasaccharides, 30% core type-1 monosialyltrisaccharides and 20% core type-2 monosialylpentasaccharides.