Complete sequencing and characterization of equine aggrecan

E. H. Caporali, T. Kuykendall, M. C. Stewart

Research output: Contribution to journalReview article

Abstract

Objectives: 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 ag-grecan sequence. Clones were sequenced and compared with the equine genome data-base 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. Clinical significance: The complete equine aggrecan sequence will support more in-depth research on aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.

Original languageEnglish (US)
Pages (from-to)79-87
Number of pages9
JournalVeterinary and Comparative Orthopaedics and Traumatology
Volume28
Issue number2
DOIs
StatePublished - Jan 1 2015

Keywords

  • Aggrecan
  • Aggrecanase
  • Cartilage
  • Matrix metallo-proteinase

ASJC Scopus subject areas

  • Animal Science and Zoology
  • veterinary(all)

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