Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung

Abstract

BackgroundMice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity. Mice express primarily three CLPs, including breast regression protein-39 (BRP-39) [chitinase 3-like-1 (Chi3l1) or 38-kDa glycoprotein (gp38k)], Ym1 (Chi3l3) and Ym2 (Chi3l4). Recently, CLPs have attracted considerable attention due to their increased expression in a number of pathological conditions, including asthma, allergies, rheumatoid arthritis and malignant tumors. Although the exact functions of CLPs are largely unknown, the significance of their increased expression levels during pathophysiological states needs to be determined. The quantification of BRP-39, Ym1 and Ym2 is an important step in gaining insight into the in vivo regulation of the CLPs.MethodsWe constructed a standard DNA for quantitative real-time PCR (qPCR) by containing three CLPs target fragments and five reference genes cDNA in a one-to-one ratio. We evaluated this system by analyzing the eight target cDNA sequences. Tissue cDNAs obtained by reverse transcription from total RNA from four embryonic stages and eight adult tissues were analyzed using the qPCR system with the standard DNA.ResultsWe established a qPCR system detecting CLPs and comparing their expression levels with those of five reference genes using the same scale in mouse tissues. We found that BRP-39 and Ym1 were abundant in the mouse lung, whereas Ym2 mRNA was abundant in the stomach, followed by lung. The expression levels of BRP-39 and Ym1 in the mouse lung were higher than those of two active chitinases and were comparable to glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene which is constitutively expressed in all tissues.ConclusionOur results indicate that catalytically inactive BRP-39 and Ym1 are constitutive genes in normal mouse lung.

Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli

“…We recently reported that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach [ 22 , 23 , 35 ]. The stomach is an important organ that plays fundamental roles in the digestion of foods and the protection against harmful organisms.…”

Comprehensive Assessment of Host Responses to 5-Fluorouracil-Induced Oral Mucositis through Transcriptomic Analysis

“…Chi3l4 is a kind of chitinase-like proteins (CLPs) in rodents, and it has been proposed that Chi3l4 participates in the processes of the carbohydrate metabolism, chitin catabolism, and inflammatory response [ 41 ]. Webb et al [ 42 ] suggested IL-4 might increase the expression of Chi3l4 mRNA in a STAT6-dependent fashion in the mouse model of ovalbumin-induced allergic airway inflammation.…”

Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

“…We previously established a real-time PCR system that is capable of quantifying mRNA expression of two mammalian chitinases in mouse tissues while comparing their levels with those of reference genes using the same scale [ 45 , 46 , 47 ]. In this study, we aimed to quantify the levels of YKL-40 (inactive protein) and compare them with expression of Chit1, AMCase (active chitinases) and housekeeping genes GAPDH and β-actin in human tissues ( Figure 1 A).…”

“…Recently, we established a quantitative real-time PCR (qPCR) system using a single standard DNA molecule to quantify the expression levels of CLPs, chitinases and reference genes [ 45 , 46 , 47 ]. This tool enables us to analyze the expression levels of multiple genes at the same scale.…”