Dataset Information

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

ABSTRACT: Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.

SUBMITTER: Jacobsson KG

PROVIDER: S-EPMC1148290 | biostudies-other | 1987 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3'-phosphate 5'-phosphosulphate.

Dataset Information

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets