Abstract
Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10−5 M−1s−1, for rSW-AT-1 and chymotrpsin is 2.85×10−6 M−1s−1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4–10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed.
Highlights
IntroductionSerpins (serine-proteinase inhibitors) are a broadly distributed family of protease inhibitors, and most of the members in this family have an intrinsic inhibitory specificity against serine proteases [1]
Serpins are a broadly distributed family of protease inhibitors, and most of the members in this family have an intrinsic inhibitory specificity against serine proteases [1]
silkworm antitrypsin (SW-AT)-1, known as SW-AT in previous studies, was reported as a antitrypsin in hemolymph of B. mori, we identified it in body wall by mass spectrometry in our previous study
Summary
Serpins (serine-proteinase inhibitors) are a broadly distributed family of protease inhibitors, and most of the members in this family have an intrinsic inhibitory specificity against serine proteases [1]. Serpins have a hyper-variable amino acid (Aa) sequence around its reactive site, usually at the Cterminus. This region formed a surface loop protruding from the body of the molecule called the reactive centre loop (RCL). Expression pattern analysis indicated that Bmserpin-2 may be involved in B. mori antiviral response [12]. Another antichymotrypsin serpin (sw-Achy) was cloned from larval fat body. Similar with the serpins in M. sexta, four splice variants were designated as SW-AT-1, SW-AT-2, SWAT-3, and SW-AT-4 These isoforms showed tissue-specific expression patterns, with SW-AT-1 present in almost all tissues and sw-AT-4 found in only a few tissues. Low temperature had the greatest effect on isoform expression, and the expression was upregulated with mycotic
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