Complete sequencing and characterization of equine aggrecan.

The researchers have completely sequenced and characterized equine aggrecan, confirming the conservation of certain cleavage sites. This research might aid further studies into aggrecan processing and degradation in equine musculoskeletal tissues.

Methodology

• The researchers used reverse transcription-polymerase chain reaction (a common laboratory technique used to make many copies of a specific DNA sequence) and rapid amplification of cDNA ends to generate clones that encompassed the complete equine aggrecan sequence.
• The clones were sequenced and compared to the equine genome database to ascertain intron-exon boundaries (points where DNA sequences are divided into protein-encoding sequences or exons and non-coding sequences or introns).

Results

• The discovered aggrecan gene spans over 61 kb on chromosome 1 and is composed of 17 exons.
• They identified two major variants of the aggrecan – one containing 8187 base pairs (bp) or 2728 amino acids, and a second sequence of 8061 nucleotides or 2686 amino acids. The variation between the two was due to a CS1 domain polymorphism (genetic variation).
• Both sequences were found to be larger than what was predicted in the genomic database. The genomic database was missing 11 CS1 repeat elements.
• The researchers compared the equine amino acid sequence with human, bovine, and murine sequences. They noted a high level of conservation in the globular domains 1, 2, and 3 (over 80% identity).
• The equine CS1 domain was found to be considerably larger than in other species and thus was the least conserved domain, with only 22% overall amino acid identity.
• They identified previously defined aggrecanase, calpain, and MMP cleavage sites. A cleavage site is a location on the protein where it can be cut.
• Western blotting was used to observe post-secretion processing in chondrocyte culture samples.

Conclusions

• The researchers concluded that their sequencing and characterization of the complete equine aggrecan sequence will aid further in-depth research, particularly concerning aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.