The structural heterogeneity of the acute phase proteins from domestic species

[摘要] The principal aims of the work presented in this thesis were to investigate the structural heterogeneity of the acute phase proteins from domestic animal species and to document the changes within the carbohydrate moiety during disease. Alpha-1-acid glycoprotein (AGP) is a positive acute phase protein in many species, with its concentration in the plasma increasing 2 to 5 fold in a number of pathophysiological states. Human AGP is extensively glycosylated (45 percent) due to 5 N linked glycan chains. In normal serum, AGP does not exist in a single form but as a heterogeneous population of glycoforms. Heterogeneity arises through subtle structural differences in monosaccharide sequence and linkages, degree of branching and extent of fucosylation and sialylation. The existence of structurally distinct glycoforms implies a functional diversity since the properties of a glycoprotein are influenced by the structures of its oligosaccharide chains. During pathological conditions in humans, not only may the total concentration of AGP be altered but the relative proportions of the normal AGP glycoforms can change and abnormal glycoforms are expressed. A method for the isolation of feline AGP from serum and ascetic fluid was developed which maintained the structural integrity of the glycan chains. Feline AGP was isolated from serum or ascitic fluid from 17 individual cats that were diagnosed to have feline infectious peritonitis (FIP), a disease known to cause an increase in AGP concentrations, and also isolated from pooled serum from animals without inflammatory disease. Following isolation of fAGP the monosaccharide composition and oligosaccharide profile was analysed by high pH anion exchange chromatography and pulsed amperometric detection. It was found that AGP from cats with FIP expressed significantly more (p 05) N-acetylglucosamine and galactose residues than AGP isolated from pools of serum from cats without FIP or other inflammatory disease. Fucose residues were present on the fAGP glycan chains of cats 5 and 9. In the presence of FIP, fAGP carried both disialylated and trisialylated glycan chains while fAGP from cats without FIP only expressed disialylated glycan chains. The structural heterogeneity of bovine AGP in bovine mastitis was examined by lectin binding with Sambucus Nigra, Concanavalin A, Maackia Amurensis or Aleuria aurantia lectins that bind a 2-6 linked sialic acid, diantennary chains, a 2-3 sialic acid and fucose residues respectively. Bovine AGP from cows suffering from mastitis was hypersialylated with more diantennary chains compared to AGP from control cows. It was also found that bovine AGP had no detectable a2-3 sialic acid or fucose residues. A novel isoform of bovine p-haptoglobin was detected in milk during bovine mastitis. Investigation using PNGase F and lectins showed that the difference between the novel isoform and liver derived form was due to glycosylation changes. The novel isoforms of pHp was 3kDa heavier than serum Hp, however following removal of N-linked glycan chains by PNGase F both forms of Hp had a molecular weight of 30kDa. Fucose was expressed on the glycan chains of the novel isoforms of pHp while no fucose residues were detectable on the serum form of pHp. Finally a comparative study looking at the glycan chain of AGP from cat, cow, sheep and man revealed distinct differences between the different species although AGP from the ruminants was more similar than AGP from man or cat. Fucose was expressed within the glycan chains of human AGP only, while only AGP from sheep expressed N- acetylgalactosamine residues. Alpha-1-acid glycoprotein from cat, cow and sheep expressed N-acetyl neuraminic acid as well as N-glycolyl neuraminic acid, while human AGP expressed N-acetylneuraminic acid only. Overall the findings detailed in this thesis indicated that the structural heterogeneity of AGP from domestic species is influenced by the disease status of the host.

[发布机构] University:University of Glasgow

[关键词] Molecular biology