Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs

• Published in: Insect biochemistry and molecular biology Vol. 42; no. 7; pp. 482 - 490
• Main Authors: Tamaki, Fábio K, Padilha, Marcelo H.P, Pimentel, Andre C, Ribeiro, Alberto F, Terra, Walter R
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2012
• Subjects: Amino Acid Sequence; Animals; antiserum; Cell membranes; Chymotrypsin; Chymotrypsin - chemistry; Chymotrypsin - genetics; Chymotrypsin - metabolism; Chymotrypsin expression; Chymotrypsin secretion; Chymotrypsin sequence; Cloning, Molecular; Digestive System - chemistry; Digestive System - enzymology; Dipteran chymotrypsins; DNA, Complementary - genetics; Drosophila melanogaster; Drosophila melanogaster - chemistry; Drosophila melanogaster - enzymology; Drosophila melanogaster - genetics; Enzymes; Escherichia coli; Evolution; Exocytosis; Gene expression; genes; half life; heat inactivation; Houseflies - chemistry; Houseflies - enzymology; Houseflies - genetics; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; Larva - chemistry; Larva - enzymology; Larva - genetics; Larvae; Membrane-bound chymotrypsin; Midgut; Migration; molecular weight; Musca; Musca domestica; pH effects; Phylogeny; polyacrylamide gel electrophoresis; Polymerase chain reaction; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; sequence analysis; Sequence Analysis, DNA; Soluble chymotrypsin; transcriptome; Vesicles; Western blotting; Chymotrypsin expression; Chymotrypsin sequence; Chymotrypsin secretion; Membrane-bound chymotrypsin; Dipteran chymotrypsins; Soluble chymotrypsin
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2012.03.005

Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46°C) in the presence and absence of 10mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila.