Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans

Abstract

Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for trypsin and chymotrypsin occurs at the post-transcriptional level in tsetse. Both the coding sequences and patterns of expression of Gsp1 and Gsp2 genes are similar to the serine proteases that have been reported from the bloodfeeding insect Stomoxys calcitrans.

Functional Characterization of HFR1, a High-MannoseN-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae

“…Levels of the digestive enzymes trypsin-and chymotrypsin-like Ser proteases are low in neonate Hessian fly larvae that have not fed on plants. But in the Avirulent biotype L larvae on resistant H9-Iris seedlings 6 16 13.1 6 1.0 (16) gut of virulent larvae, the levels of these digestive enzymes increase over time (Shukle et al, 1985;Zhu et al, 2005), just as in the gut of another dipteran, the tsetse fly (Glossina morsitans), in which Ser proteases are induced by feeding (Yan et al, 2001). An induction of digestive enzymes may be responsible for the differing banding patterns of HFR1 proteolytic products observed in immunodetection experiments with virulent and avirulent larvae.…”

A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm

“…CTLP1 meets all essential structural requirements of digestive serine proteases, as it possesses conserved histidine, aspartate and serine residues forming the catalytic triad (Kraut, 1977;Law et al, 1977). Moreover, it contains six cysteine residues typically present in invertebrate serine proteases (Fig.·2) (Yan et al, 2001). The presence of the three amino acids glycine, glycine and aspartate in the primary specificity pocket suggests that CTLP1 may exhibit a chymotrypsin-like substrate specificity with a glycine at the bottom of the pocket as the primary determinant (Perona and Craik, 1997).…”

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae

“…Biochemical characteristics of proteolytic enzymes including carboxypeptidase A, carboxypeptidase B, aminopeptidase, and members of the serine protease family (including a trypsin-like enzyme, a chymotrypsin-like enzyme, and trypsin) have been widely studied (1). These major digestive enzymes play important roles in protein digestion and absorption (2).…”