Purification and assays of tachylectin-5

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250 mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004–0.008 μg/mL for tachylectin-5A and 0.077–0.27 μg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3–1.6 mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04–0.08 μg/mL for tachylectin-5A and 0.05–0.11 μg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3–2.4 μg/mL for tachylectin-5A and 15.1–26.8 μg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages277-283
Number of pages7
DOIs
Publication statusPublished - 2020

Publication series

NameMethods in Molecular Biology
Volume2132
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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