Complete primary sequence of equine cartilage link protein deduced from complementary DNA.

This research focused on understanding the structure of equine link protein (LP) found in the cartilage, how it varies with age, and its similarity to LP in other species. It recognized three distinct forms of LP and found that two forms tend to accumulate with age. The research then sequenced the protein, finding a high degree of similarity to the sequence in humans and other species. This suggests that LP’s creation and processing likely happens in the same way across a variety of species.

Understanding the Structure of Equine Link Protein

• The research studied the structure of link protein (LP) in equine articular cartilage sampled from horses of various ages ranging from 1 to 15 years.
• Three distinct isoforms of the protein were identified. Isoforms are versions of a protein that are similar but not identical. These isoforms had different molecular weights: 46,000, 43,000, and 41,000.
• The relative amounts of these isoforms were found to change with the age of the horse. The largest form remained constant throughout all ages; however, the smaller two forms (43,000 and 41,000) were found to increase with the horse’s age.
• This led researches to believe that these proteins were accumulating in the extracellular matrix of the cartilage because of proteolytic cleavage, which is a process where proteins are broken down into smaller peptides.

Deducing the Complete Primary Sequence from Complementary DNA

• To fully understand the protein, the researchers determined the complete amino acid sequence of the protein core. This sequence was deduced from complementary DNA products that were prepared by amplifying the cartilage LP mRNA through a polymerase chain reaction.
• When compared, the sequence of the equine LP was found to be 96% identical to that of humans and other species where the primary structure of LP has been determined. This great similarity indicates that the mechanisms involved in the extracellular processing of LP are most likely comparable across several species.

Analysis of LP at the Transcription Level

• The research also assessed LP expression at the transcription level; essentially, how the protein is created from DNA.
• Equine chondrocytes (cartilage cells) were found to express LP as two abundant mRNA forms of sizes 5.0 and 3.0 kb, along with a smaller mRNA form of size 1.5 kb.
• The processing of LP mRNA in horses appears to be similar to that observed in other species, suggesting that while there are multiple transcript versions, the coding region retains its consistency and results in the formation of only one protein product.