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.

Misa Ohno,Fumitaka Oyama,Yuta Kida,Yasusato Sugahara,Masayoshi Sakaguchi

doi:10.1186/1471-2199-15-23

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.

Highlights

Mice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity
Our study shows that the expression levels of breast regression protein-39 (BRP-39) and Ym1 in the mouse lung are higher than those of two active chitinases and are comparable to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene which is constitutively expressed in all tissues to maintain cellular functions [42,43,44]
We established and validated a quantitative real-time PCR (qPCR) system for individual quantification of the expression of three CLPs and comparing their expression levels with those of reference genes using the same scale in mouse tissues

Summary

Introduction

Mice 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). Mice express primarily breast regression protein-39 (BRP-39) [chitinase 3-like-1 (Chi3l1) or 38-kDa glycoprotein (gp38k)], Ym1 (Chi3l3) and Ym2 (Chi3l4), whereas humans produce YKL-40 (CHI3L1 or human cartilage glycoprotein-39), the human homologue of BRP-39, but do not synthesize Ym1 and Ym2 [3,4,5,6,7,8,9,10]. Ym1 shows a high degree of sequence homology to Ym2 with 91% amino acid sequence identity, but these proteins exhibit different expression patterns [5,6,9]

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