Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Abstract

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with 35S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and 35SO 4 2−, mutant cartilage extracts showed lower incorporation of 35SO 4 2− into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of 35S]PAPS and of 35S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.