Measurement of Chitinase Activity in Biological Samples

Summary

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Abstract

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

Introduction

Chitin is the second most abundant polysaccharide on the earth after cellulose, serving as a major structural component of a variety of organisms including the exoskeleton of insects, fungi, yeast, and algae; some vertebrates also have chitin¹. Chitinases are a family of enzymes that are capable of breaking down chitin and are highly conserved throughout evolution in species ranging from bacteria to mammals^2,3. In addition to chitinases, mammals also have chitinase-like proteins which are similar to chitinases in their ability to bind chitin but differ in that they lack enzymatic abilit....

Protocol

All animal procedures were performed under an IACUC-approved protocol at Yale University School of Medicine.

1. Mouse Sample Collection

1. Anesthetization
   1. Anesthetize mice using ketamine and xylazine (100 mg/kg of ketamine and 10 mg/kg of xylazine).
2. Blood sample collection
   1. Confirm the depth of anesthesia by non-responsiveness to a toe pinch.
   2. Surgically open the chest cavity to expose the heart.
   3. Insert a 26.5 G needle into the left ventricle and collect blood into a syringe.
      NOTE: Typically, about 0.5̵....

Results

The results shown here are from a study measuring the chitinase activity in serum and BAL samples of wild type (C57BL/6) and Chit1 transgenic mice (on C57BL/6 background) that overexpress the Chit1 gene on a doxycycline promoter. Our data show that both serum and BAL samples have detectable chitinase activity at baseline 698.2 ± 189.9 nmol/mL·h and 485.7 ± 114 nmol/ml·h, respectively. Overexpression of the Chit1 gene using doxycycline in drinking water for 4 weeks resulted in the elevation of chitinase activity that was .......

Discussion

Chitinase activity has emerged as an important biomarker for predicting disease severity, disease progression, therapeutic effectiveness and the presence of specific pathogens¹⁸. Although many of the long-postulated theories about the role of chitinases have not been experimentally proven¹⁹, new studies have provided important insights into the role of chitinases and chitinase like proteins in various diseases^2,9

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-methylumbelliferone	Sigma	M1381	Standard: commonly used in flourimetric assays for determination of enzyme activity
4MU-GlcNAc2	Sigma	M9763	fluorescent chitinase substrate for use in mouse samples
4MU-GlcNAc3	Sigma	M5639	fluorescent chitinase substrate for use in human samples
Citric Acid-monohydrate			for use in McIlvain Buffer
Glycine			for use in Stop Buffer at a concentration of 0.3 M
Na2HPO4xH2O			for use in McIlvain Buffer
NaOH			for use in Stop Buffer at a concentration of 12 g/L
Vision Plate: Non-sterile, untreated black 96 well plate	4titude	4ti-0224