Abstract
Murine erythroleukemia cells (Friend erythroleukemia cells of a C-10-6 line) synthesized sulfated glycosaminoglycans consisting mainly of heparan sulfate (more than 95%) with a small amount of chondroitin 4-sulfate. The heparan sulfate occurred as proteoglycans, of which the cell-associated component was separated into urea-insoluble (UI) and urea-soluble (US) fractions. The UI proteoglycan consisted of a single homogeneous molecular species with an estimated Mr of 360,000 (C(UI)PG), whereas the US component was composed of two subfractions: a homogeneous species with an Mr of 280,000 (C(US)PGI) and a mixture of compounds with Mr values of less than 80,000 (C(US)PGII), which were isolated in yields of about 110, 340, and 80 micrograms of hexuronate (HexUA), respectively, from 1.37 g of an acetone powder prepared from 5.7 x 10(9) cells in the logarithmic phase of growth. The proteoglycan released into the medium (12 liters) was a single homogeneous species with an Mr of 320,000 (MPG) which was purified in a yield of 500 micrograms of hexuronate. The major, cell-associated proteoglycan, C(US)PGI, had very high contents of serine and glycine, accounting for approximately 80% of the total amino acids. This proteoglycan as well as the other two large proteoglycans, C(UI)PG and MPG, were highly resistant to degradation by various proteinases. These three proteoglycans, C(UI)PG, C(US)PGI, and MPG, had heparan sulfates with estimated Mr values of 32,000, 27,000, and 30,000. On the other hand, the Mr of the smaller proteoglycan, C(UI)PGII, was not significantly different before and after beta-elimination, indicating that it contains only a small peptide, if any. The heparan sulfate of this proteoglycan consisted of smaller and heterogeneous molecular species with Mr values of 26,000, 20,000, and 4,000. Digestion of these heparan sulfates with heparitinase I plus II resulted in almost complete depolymerization and gave six unsaturated disaccharides, delta HexUA-GlcNAc, delta HexUA-Glc-NAc(6-SO4), delta HexUA-GlcNSO3, delta HexUA-GlcNSO3 (6-SO4), delta HexUA(2-SO4)-GlcNSO3, and delta HexUA(2-SO4)-GlcNSO3(6-SO4). The relative amounts of these disaccharides generated from the individual heparan sulfates showed that an average ratio of sulfate residues to repeating disaccharide units of the C(US)PGII-derived heparan sulfate (0.97) was significantly higher than those of the other three large proteoglycan-derived glycosaminoglycans (0.54-0.70).
Highlights
Kemia cells of a (2-10-6 line) synthesized sulfated gly- derived heparan sulfat(e0.97) was significantlyhigher cosaminoglycansconsisting mainly of heparan sulfate than those of the other three large proteoglycan-de
The UI proteoglycan consisted of a single homogeneous molecular species with an estimated M, of 360,000 (C(UI)PG),whereas theUS component was composed of two subfractions: a homogeneous species with an M, of 280,000 (C(US)PGI)and a mixture of compounds with M, values of less than 80,000 (C(US)PGII), which were isolated in yields of about
The proteoglycan released into the medium (12 liters) wasa single homogeneous species with an M, of 320,000 (MPG) which was purified in a yield of 500
Summary
Cells cans produced bycells of other hemopoietic lineagesF. or this purpose wechose murine erythroleukemiacells (Frienderythroleukemia cells). ~-threo-hex-4-enopyranosyluroniacid)-6-O-sulfo-D-glucos(eADiHStriS), which were produced by digestion of heparan sulfates with a combination of heparitinases I and 11, were prepared as described previously [33].The molecular weight standards used on gel filtration. Proteoglycans of Murine Erythroleukemia Cells were washedthree times with the medium without fetal bovine serum and the isotope and extracted with 15 ml of 4 M guanidine HC1, 50 mM Tris-HC1,pH 7.3, containing 10 mM EDTA, 10mM N-ethylmaleimide, 1mM phenylmethylsulfonyl fluoride, and 0.036 mM pepstatin A as proteinase inhibitors for 24 h on ice with continuous stirring. The radiolabeled and unlabeled extracts were combined and dialyzed against five changes of 4 liters of 4 M urea, 20 mM NaC1, 20 mM Tris-HC1,pH 7.3, on ice. The insoluble material appearing during dialysis was collected by centrifugation at 8,000 X g for 20 min, washed twice with dialyzing buffer, and dissolved in the extraction buffer. Deaminative degradation of glycosaminoglycansby nitrous acid treatment was carried out as described previously [47]
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