Identification and characterization of a Leishmania donovani serine protease inhibitor: Possible role in regulation of host serine proteases

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• 作者：Md Nur Alam ; Partha Das ; Tripti De ; Tapati Chakraborti ; ^{t_chakra08@rediffmail.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：BApNA ; N-α-benzoyl&ndash ; DL-arginyl-p-nitroanilide ; BSA ; bovine serum albumin ; CBB ; Coomassie brilliant blue ; ICP ; cysteine protease inhibitor ; ISPs ; serine protease inhibitors ; LdISP ; Leishmania donovani serine protease inhibitor ; MS ; mass spectrometry ; PBS ; phosphate-buffered saline ; PMF ; protein mass fingerprinting ; SP-Ld ; Leishmania donovani serine protease
• 刊名：Life Sciences
• 出版年：2016
• 出版时间：1 January 2016
• 年：2016
• 卷：144
• 期：Complete
• 页码：218-225
• 全文大小：1406 K

文摘

This study aims to identify, purify, and characterize an endogenous serine protease inhibitor from an Indian strain of Leishmania donovani, which causes the fatal visceral leishmaniasis.

Main methods

(i) Reverse zymography was used to identify the serine protease inhibitor by inhibiting the gelatinolytic activity of serine protease. (ii) Purification was performed by combining heat treatment, ultracentrifugation, and affinity and gel permeation chromatography. (iii) Spectrophotometric assays were conducted to quantify and compare the inhibitory activity of the L. donovani serine protease inhibitor (LdISP). (iv) Further, the protein was identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS).

Key findings

An endogenous inhibitor with an apparent molecular weight of 21.8 kDa, which is acidic in nature, having a pI of 5.9 was identified. The K_i value of the inhibitor for trypsin was determined to be in the nanomolar range. The protein has the following features: (i) ecotin-like nature, (ii) cross-organism functionality, that is, an inhibitory effect on the serine proteases of higher organisms other than its own, and (iii) homology with other such proteins from a different species of Leishmania on conducting protein mass fingerprinting after MALDI ToF MS.

Significance

The inhibitor shows varying and entirely contrasting efficacies toward serine proteases of its own as well as of higher organisms. This indicates that it accelerates disease progression and drives parasite survival as it inhibits the activities of the host serine proteases.