Complete sequencing and characterization of equine aggrecan

E. H. G. Caporali; T. Kuykendall; M. C. Stewart

doi:10.3415/VCOT-14-05-0069

Veterinary and Comparative Orthopaedics and Traumatology, Table of Contents

Vet Comp Orthop Traumatol 2015; 28(02): 79-87
DOI: 10.3415/VCOT-14-05-0069

Original Research

Schattauer GmbH

Complete sequencing and characterization of equine aggrecan

E. H. G. Caporali

¹Department of Veterinary Clinical Medicine, University of Illinois, Champaign-Urbana, IL, USA

,

T. Kuykendall

¹Department of Veterinary Clinical Medicine, University of Illinois, Champaign-Urbana, IL, USA

,

M. C. Stewart

¹Department of Veterinary Clinical Medicine, University of Illinois, Champaign-Urbana, IL, USA

› Author Affiliations

Abstract

Summary

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 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.

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.

Keywords

Aggrecan - aggrecanase - matrix metallo proteinase - cartilage

Full Text

References

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