Diabetic BB/Wor rat haptoglobin exhibits a probable structural abnormality in Asn-linked oligosaccharides

Abstract

In this study we demonstrate that haptoglobin, a serum glycoprotein secreted by the liver, has altered structure in the BB/Wor diabetic rat. SDS-PAGE of haptoglobin (a tetramer composed of two glycosylated β-chains each containing two sites for Asn-linked oligosaccharides connected by disulfide bonds with two nonglycosylated α-chains) clearly shows that the β-chain of haptoglobin from diabetic rats is smaller than normal, with a molecular mass of 39 instead of 40 kDa. Both acute and chronic diabetic rats exhibit the defect. Defective haptoglobin appears in the serum within 4 days of onset of the disease, but insulin therapy prevents the defect. Removal of Asn-linked oligosaccharides with peptide: N-glycosidase F from Flavobacterium meningosepticum abolished the size difference between the β-chains from normal and diabetic haptoglobin, with the molecular mass in both cases shifting to 30 kDa. Haptoglobin from both normal and diabetic rats was resistant to digestion by endoglycosidase H from Streptomyces griseus, which cleaves high mannose-type chains. Removal of sialic acid with neuraminidase treatment resulted in a reduction in the molecular mass in both cases, but without eliminating the size difference between the two. These results demonstrate that haptoglobin from diabetic BB/Wor rats contains a structural abnormality which correlates with onset of the disease. The defect is most likely due to an alteration in Asn-linked oligosaccharides, probably involving a change in the neutral sugars of complex-type oligosaccharide chains. This finding represents the first example of an altered Asn-linked oligosaccharides in diabetes.