Scott P G, Nakano T, Dodd C M
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochim Biophys Acta. 1997 Aug 29;1336(2):254-62. doi: 10.1016/s0304-4165(97)00040-8.
Pig knee menisci were dissected into three zones of equal width representing inner, i.e. medial (zone 1), middle (zone 2) and outer, i.e. lateral (zone 3) tissue. Proteoglycans (PGs) were extracted with guanidinium chloride, isolated by ion-exchange chromatography and separated into two groups ('small' and 'large') by gel filtration. The small PGs were further fractionated by hydrophobic-interaction chromatography on Octyl-Sepharose. The PG eluting earliest from Octyl-Sepharose was identified as decorin on the basis of the size of the protein core produced by digestion with chondroitinase ABC, its recognition by monoclonal antibodies raised against bovine decorin and its N-terminal sequence, 23 of 24 amino acids of which were identified. Decorin represented about 23%, 28% and 32% of the total small PG recovered from Octyl-Sepharose from zones 1, 2 and 3, respectively. The major small PG in the meniscus, eluting from Octyl-Sepharose after decorin, was identified as biglycan by the size of core, recognition by a polyclonal antiserum raised against bovine biglycan and sequence of the N-terminal 26 amino acids. Biglycan accounted for approximately 53%, 52% and 38% of PG recovered from zones 1, 2 and 3, respectively. The glycosaminoglycan chains on both decorin and biglycan were identified as dermatan sulphate by their susceptibility to chondroitinase-B. Stains-All staining of SDS gels of Octyl-Sepharose eluates revealed the presence of a third small PG eluting slightly later than biglycan. This PG was purified by a further cycle of chromatography on Octyl-Sepharose and identified as fibromodulin on the basis of its amino acid composition and the N-terminal sequence obtained after digestion with pyroglutamate aminopeptidase. It was obtained in highest amounts from the inner (zone 1) tissue, which also yielded more biglycan and less decorin. Fibromodulin from the meniscus was shown to inhibit the formation of fibrils from a solution of type I collagen, independently of the effects of decorin. These results support the concept that the distributions and characteristics of the small PGs in knee meniscus reflect regional adaptation to functional demands.
猪膝关节半月板被切成三个等宽区域，分别代表内侧（即内侧，区域1）、中间（区域2）和外侧（即外侧，区域3）组织。蛋白聚糖（PGs）用氯化胍提取，通过离子交换色谱法分离，并通过凝胶过滤法分为两组（“小”和“大”）。小PGs通过辛基琼脂糖上的疏水相互作用色谱进一步分级分离。根据用软骨素酶ABC消化产生的蛋白核心大小、针对牛核心蛋白聚糖产生的单克隆抗体的识别及其N端序列（其中24个氨基酸中的23个已被鉴定），最早从辛基琼脂糖上洗脱的PG被鉴定为核心蛋白聚糖。核心蛋白聚糖分别占从区域1、2和3的辛基琼脂糖中回收的总小PG的约23%、28%和32%。半月板中的主要小PG，在核心蛋白聚糖之后从辛基琼脂糖上洗脱，根据核心大小、针对牛双糖链蛋白聚糖产生的多克隆抗血清的识别以及N端26个氨基酸的序列，被鉴定为双糖链蛋白聚糖。双糖链蛋白聚糖分别约占从区域1、2和3回收的PG的53%、52%和38%。核心蛋白聚糖和双糖链蛋白聚糖上的糖胺聚糖链因其对软骨素酶B的敏感性而被鉴定为硫酸皮肤素。辛基琼脂糖洗脱液的SDS凝胶的全染色显示存在第三种小PG，其洗脱时间略晚于双糖链蛋白聚糖。这种PG通过在辛基琼脂糖上的进一步色谱循环纯化，并根据其氨基酸组成和用焦谷氨酸氨肽酶消化后获得的N端序列鉴定为纤调蛋白。它从内侧（区域1）组织中获得的量最高，该组织也产生更多的双糖链蛋白聚糖和更少的核心蛋白聚糖。半月板中的纤调蛋白被证明可以抑制I型胶原溶液中纤维的形成，与核心蛋白聚糖的作用无关。这些结果支持了这样的概念，即膝关节半月板中小PGs的分布和特征反映了区域对功能需求的适应性。
