Goldberg H A, Domenicucci C, Pringle G A, Sodek J
Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
J Biol Chem. 1988 Aug 25;263(24):12092-101.
To provide a more definitive characterization of the hydroxylapatite-associated proteoglycans (HAPG) of bone, proteins were extracted from the mineralized matrix of fetal porcine calvaria with 0.5 M EDTA in the absence of guanidine HCl. The small proteoglycans obtained in the extract were fractionated by gel filtration on Sepharose CL-6B, purified by ion-exchange chromatography on Polyanion matrix (fast protein liquid chromatography), and then separated into three major populations of chondroitin sulfate proteoglycans by chromatography on hydroxylapatite, all in the presence of 7 M urea. Based on immunological and chemical properties, two classes of bone proteoglycan were resolved. In one class (HAPG1), the proteoglycan and specific CNBr-derived peptides cross-reacted with three monoclonal antibodies that recognize different epitopes of the protein core of bovine skin proteodermatan sulfate. The other class of proteoglycan included two species (HAPG2, HAPG3) which were not recognized by these antibodies. In addition, these proteoglycans did not stain with Coomassie Blue R-250 nor with silver stain nor did they bind to nitrocellulose membranes used in Western blots. However, the cationic dye Stains-all stained both HAPG2 and HAPG3; the protein cores of these proteoglycans were stained a characteristic turquoise blue, whereas the protein core of HAPG1 was stained pink. The average Mr values of the bone proteoglycans, from gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis were: HAPG1, 120,000, with a protein core (chondroitinase AC-digested) of 45,000; HAPG2 and HAPG3, 110,000, with protein cores of 37,000-38,000. On 15% polyacrylamide gel electrophoresis, the protein cores of HAPG2 and HAPG3 migrated with an Mr 30,000, while HAPG1 protein core was unchanged (Mr 45,000). Based on amino acid analysis, the protein chains of HAPG2 and HAPG3 appear to be identical, although minor differences in the relative amount of glucosamine were evident. In contrast, the composition of HAPG1 was quite different, with higher relative amounts of hydrophobic and aromatic residues and lower amounts of Asx and Glx. The presence of 360 residues/1,000 of Asx and Glx in HAPG2 and HAPG3 may in part explain the characteristic staining and immunotransfer properties of these proteoglycans. The unique amino-terminal sequence of HAPG2 (Asn-Pro-Val-Ala-Arg-Tyr-Gln), together with the immunological and chemical properties, would indicate that HAPG2 and HAPG3 are novel proteoglycans and, unlike HAPG1, could be unique to mineralized tissues.
为了更确切地描述骨中与羟基磷灰石相关的蛋白聚糖（HAPG），在没有盐酸胍的情况下，用0.5M乙二胺四乙酸（EDTA）从胎猪颅骨的矿化基质中提取蛋白质。提取物中获得的小蛋白聚糖通过在琼脂糖凝胶CL-6B上进行凝胶过滤分级分离，通过在聚阴离子基质上进行离子交换色谱（快速蛋白质液相色谱）纯化，然后在7M尿素存在下通过在羟基磷灰石上进行色谱分离为三个主要的硫酸软骨素蛋白聚糖群体。基于免疫和化学性质，解析出两类骨蛋白聚糖。在一类（HAPG1）中，蛋白聚糖和特定的溴化氰衍生肽与三种单克隆抗体发生交叉反应，这三种单克隆抗体识别牛皮肤蛋白聚糖硫酸皮肤素蛋白核心的不同表位。另一类蛋白聚糖包括两种（HAPG2、HAPG3），这些抗体不能识别它们。此外，这些蛋白聚糖不能用考马斯亮蓝R-250染色，也不能用银染染色，它们也不与蛋白质印迹中使用的硝酸纤维素膜结合。然而，阳离子染料“全染剂”能使HAPG2和HAPG3都染色；这些蛋白聚糖的蛋白核心被染成特征性的蓝绿色，而HAPG1的蛋白核心被染成粉红色。通过梯度十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得的骨蛋白聚糖的平均分子量为：HAPG1为120,000，其蛋白核心（经软骨素酶AC消化）为45,000；HAPG2和HAPG3为110,000，其蛋白核心为37,000 - 38,000。在15%聚丙烯酰胺凝胶电泳上，HAPG2和HAPG3的蛋白核心以分子量30,000迁移，而HAPG1蛋白核心不变（分子量45,000）。基于氨基酸分析，HAPG2和HAPG3的蛋白质链似乎相同，尽管氨基葡萄糖的相对量存在细微差异。相比之下，HAPG1的组成有很大不同，疏水和芳香族残基的相对量较高，而天冬氨酸（Asx）和谷氨酸（Glx）的量较低。HAPG2和HAPG3中每1000个残基中有360个Asx和Glx残基，这可能部分解释了这些蛋白聚糖的特征性染色和免疫转移特性。HAPG2独特的氨基末端序列（天冬酰胺 - 脯氨酸 - 缬氨酸 - 丙氨酸 - 精氨酸 - 酪氨酸 - 谷氨酰胺），连同其免疫和化学性质，表明HAPG2和HAPG3是新型蛋白聚糖，并且与HAPG1不同，可能是矿化组织所特有的。
