Isolation and characterization of small proteoglycans from different zones of the porcine knee meniscus.
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.
Scott
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PG
Department of Biochemistry, University of Alberta, Edmonton, Canada. Scottp@gpu.srv.ualberta.ca
Nakano
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T
Dodd
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CM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D008592:Menisci, Tibial / Q000737:chemistry*; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D013552:Swine
D000596:Amino Acids; D011509:Proteoglycans