Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity

Abstract

Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.

The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases

“…However, the role that these duodenases play in the digestion of food for ruminants is largely unexplored. We have recently shown that these duodenases, which are highly homologous in sequence, have become enzymes with very diverse both primary and extended cleavage specificities, indicating several different biological targets [31]. Based on a screening of the total proteome of cattle or sheep using the consensus sequences of the extended cleavage specificity of two of these enzymes, we found that mucin-5B, the major salivary mucin, is one candidate substrate for the bovine enzyme, and several chemokine receptors are candidate substrates for the sheep enzyme.…”

“…The majority of new genes belong to a new subfamily of proteases named duodenases due to their tissue specificity. They are no longer expressed in hematopoietic cells but in the duodenum, where they most likely take part in food digestion [23,31]. There is one such duodenase gene in the pig locus, four in cattle and five in sheep (Figure 1).…”

“…In a recent study we have looked into the new subfamily of chymase-locus-encoded proteases in ruminants and pigs, the duodenases [31]. These proteases are most likely a result of a set of gene duplications originating from a cathepsin G or a granzyme gene (Figure 1).…”

“…Six of the duodenases were analyzed through either phage displays or recombinant substrates and we found that they showed a surprisingly broad panel of different specificities (Figure 8). One of the analyzed duodenases was a highly specific asp-ase (bovine MCP1A); two were highly specific tryptases (sheep MCP3 and MCP3-like); two enzymes exhibited dual specificity, being both tryptases and chymases (bovine DDN1 and DDN1-like); and one enzyme was a pure chymase (pig MCP3-like) with a relatively broad extended specificity (Figure 8) [31]. Notably, these six duodenases are all highly homologous in their primary sequence despite their large differences in primary and extended specificity, illustrating that the enzyme specificity can be changed quite dramatically by only minor changes in the primary sequence.…”

“…highly specific tryptases (sheep MCP3 and MCP3-like); two enzymes exhibited dual specificity, being both tryptases and chymases (bovine DDN1 and DDN1-like); and one enzyme was a pure chymase (pig MCP3-like) with a relatively broad extended specificity (Figure 8) [31]. Notably, these six duodenases are all highly homologous in their primary sequence despite their large differences in primary and extended specificity, illustrating that the enzyme specificity can be changed quite dramatically by only minor changes in the primary sequence.…”