title: Complete sequencing and characterization of equine aggrecan.
abstract: OBJECTIVE
  To fully sequence and characterize equine aggrecan and confirm conservation of major aggrecanase, calpain and matrix metalloproteinase (MMP) cleavage sites.
  
  
  METHODS
  Reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends were used to generate clones that encompassed the complete equine aggrecan sequence. Clones were sequenced and compared with the equine genome database to determine intron-exon boundaries.
  
  
  RESULTS
  The aggrecan gene spans over 61 kb on chromosome 1 and is encoded by 17 exons. Two major variants of aggrecan were cloned; one containing 8187 bp (2728 amino acids) and a second sequence of 8061 nucleotides (2686 amino acids). The variation was due to a CS1 domain polymorphism. Both sequences are substantially larger than predicted by the genomic database; 11 CS1 repeat elements are absent in the database sequence. The equine amino acid sequence was compared with human, bovine and murine sequences. Globular domains 1, 2 and 3 are highly conserved (overall identity over 80%). Equine CS1 is considerably larger than in other species and, therefore, is the least conserved domain (an overall amino acid identity of 22%). Previously defined aggrecanase, calpain and MMP cleavage sites were identified. Western blotting of chondrocyte culture samples showed complex post-secretion processing.
  
  
  CONCLUSIONS
  The complete equine aggrecan sequence will support more in-depth research on aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.
authors: E H G
authors: EH
authors: Caporali
authors: T
authors: T
authors: Kuykendall
authors: Matthew Stewart, VTH-UIUC, 1008W Hazelwood Drive, Urbana, IL 61802, United States, Phone: +1 217 265-5206, Fax: +1 217 244-1475, E-mail: matt1@illinois.edu.
authors: M C
authors: MC
authors: Stewart
mesh_terms: D053674:Aggrecans / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D006736:Horses / Q000502:physiology*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
chemical_list: D053674:Aggrecans; D018076:DNA, Complementary; D010450:Endopeptidases; C083641:aggrecanase
keywords: Aggrecan; aggrecanase; cartilage; matrix metallo-proteinase