Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.