Klein D J, Brown D M, Oegema T R
J Biol Chem. 1986 Dec 15;261(35):16636-52.
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.
对大鼠肾小球硫酸乙酰肝素（HS）和硫酸皮肤素（DS）蛋白聚糖的合成进行了体外和体内研究。[35S]硫酸盐掺入大分子在体外16小时内呈线性，且DS是主要的糖胺聚糖（GAG），而在体内孵育中HS占主导。蛋白聚糖存在于CsCl梯度底部的2/5（高密度）部分，并从DEAE - Sephacel柱上洗脱为两个重叠峰。低密度35S - 糖蛋白和35S - 蛋白聚糖的比例随时间增加。从肾小球中提取出两种高浮力密度的HS蛋白聚糖，并在DEAE峰I中洗脱。第一种，HS - tIA，Mr为130×10³，带有Mr为12.5×10³的GAG链。这种蛋白聚糖可被胰蛋白酶从组织中释放出来，并可被肝素处理部分取代。此外，在标记追踪实验的培养基中它能快速释放，之后在凝胶中迁移速度比HS - tIA略快，其HS链长度与其组织对应物相似。第二种，HS - tIB，Mr为8.6×10³，几乎没有或没有附着蛋白。这种蛋白聚糖被鉴定为细胞内的，因为它能抵抗胰蛋白酶处理或在培养基中肝素提取的释放，并且在标记追踪实验的培养基中未检测到。对两种组织DS蛋白聚糖进行了鉴定。第一种，DS - tIA，与HS - tIA共纯化，是体外4小时孵育期间合成的主要蛋白聚糖。与HS - tIA一样，它能快速释放到培养基中，并可被肝素或胰蛋白酶处理从细胞表面或组织“受体”上取代。第二种，来自DEAE峰II的Sepharose CL - 6B排阻的DS蛋白聚糖，DS - tII，在体外16小时内在组织中积累。这种蛋白聚糖是自缔合的，并且沿着其Mr为26×10³的DS链含有艾杜糖醛酸残基簇。它能抵抗肝素、胰蛋白酶和去污剂从组织中提取。在孵育培养基中未检测到DS - tII。相反，培养基中的蛋白聚糖以单个Sepharose CL - 6B包含峰的形式洗脱。培养基中蛋白聚糖的DS链较短（Mr为18×10³），并且与DS - tII的GAG相比，艾杜糖醛酸残基的间距更规则。DS - mII GAG的长度和硫酸化模式与DS - tIA的GAG相似。因此，肾小球快速合成并释放Sepharose CL - 6B包含的可被肝素取代的DS和HS蛋白聚糖，同时保留一种Sepharose CL - 6B排阻的自缔合DS蛋白聚糖和一种细胞内HS。
