Abstract
Rat serosal mast cells, which synthesize only heparin proteoglycans as detected by intrinsic labeling with [35S]sulfate, were analyzed for the presence of intracellular chondroitin sulfate proteoglycans by chemical and immunochemical means. Rat serosal mast cells of greater than 99% purity were treated with Zwittergent 3-12 and 4 M guanidine HCl, and the extracted nonradiolabeled proteoglycans were purified by density gradient centrifugation. As assessed by quantification of the unsaturated disaccharides released from the proteoglycans by chondroitinase ABC treatment, 10(6) rat serosal mast cells contained 2.4-4.5 micrograms of chondroitin sulfate proteoglycans. Analysis of the chondroitinase ABC digests by high performance liquid chromatography revealed the unsaturated disaccharides delta Di-4S, delta Di-diSB, and delta Di-diSE which were derived from GlcA----GalNAc-4-SO4, iduronic acid-2-SO4----GalNAc-4-SO4, and GlcA----GalNAc-4,6-diSO4, respectively. The molar ratio of the monosulfated to disulfated disaccharides was approximately 2:1 with delta Di-diSE greater than delta Di-diSB. When analyzed with a mouse anti-chondroitin sulfate monoclonal antibody and fluorescein-labeled F(ab')2 goat anti-mouse IgG, approximately 91% of permeabilized and chondroitinase ABC-treated cells in the mast cell preparations exhibited intracellular fluorescence, and the pattern of staining indicated that the chondroitin sulfate molecules were located in the secretory granules. The specificity of the monoclonal antibody for the unsaturated double bond created by chondroitinase ABC treatment of the proteoglycan in situ was established by the absence of fluorescence when the chondroitinase ABC step was omitted or when heparinase digestion was substituted for chondroitinase ABC. Furthermore, the ability of the anti-chondroitin sulfate monoclonal antibody to mediate fluorescence in situ was markedly reduced by absorption with solid-phase chondroitin sulfate proteoglycan that had been chondroitinase ABC-treated, but not by absorption with undigested proteoglycan or with solid-phase heparin. The highly sulfated chondroitin sulfate proteoglycans of rat serosal mast cells are the same type synthesized by the rat mucosal mast cell subclass.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the $Department of Medicine, Haruard Medical School and the Department of Rheumatology and Immunology, Brigham and Women’s Hospital, sulfate-containingproteoglycan is a remnant of an earlier stage of differentiation and implies an ontologic relationship between the twomast cell subclasses
When the chondroitin sulfate proteoglycans from rat mucosal mast cells are incubated with chondroitinase ABC and the generated unsaturated disaccharides are analyzed by high performance liquid chromatography (HPLC’), thethreedisaccharides ADi-4S, ADi-diSB, and ADi-diSE are detected with the concentration of ADi-4S = ADi-diSB> ADi-diSE. rat serosal mast and fluorescein-labeled F(ab’)a goat anti-mouse IgG, cells do not synthesize detectable levels of 35S-labeledchon
We demonstrate by chemical techniques that rat serosal mast cells havechondroitinsulfate proteoglycans and that their chondroitin sulfatgelycosaminoglycans contain the same types of unique disulfated disaccharides that are detected by “S-labeling of rat mucosal mast cells
Summary
Rat serosal mast cells, which synthesizeonly heparin identified by the differences in the typesof proteoglycans (1, proteoglycans as detected by intrinsic labeling with 2) and serine proteases [3, 4] that they synthesize and store [”’S]sulfate,were analyzed for the presence of intra- in their secretorygranules, bytheir differential histochemical cellular chondroitin sulfateproteoglycans by chemical staining properties [5], by their relative histamine content and immunochemical means. When the chondroitin sulfate proteoglycans from rat mucosal mast cells are incubated with chondroitinase ABC and the generated unsaturated disaccharides are analyzed by high performance liquid chromatography (HPLC’), thethreedisaccharides ADi-4S, ADi-diSB, and ADi-diSE are detected with the concentration of ADi-4S = ADi-diSB> ADi-diSE. rat serosal mast and fluorescein-labeled F(ab’)a goat anti-mouse IgG, cells do not synthesize detectable levels of 35S-labeledchon-. Two ml of chondroitin sulfate proteoglycan-Sepharose affinity resin were suspended in enriched-Trisbuffer with protease inhibitors [17] and incubated with 1 unit of chondroitinase ABC for 30 min at 37"C; the digestion was stopped by washing the gel with HBSS. The supernatants were recovered by centrifugation and the capacities of serial dilutions of these supernatants to exhibit intracellular immunofluorescent staining of permeabilized, chondroitinase ABC-treated rat serosal mast cells were compared to each other and to similar dilutions of unabsorbed antibody
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
