The authors have declared that no competing interests exist.
Conceived and designed the experiments: MS YS FO. Performed the experiments: MO KT FO. Analyzed the data: MO KT. Contributed reagents/materials/analysis tools: MO KT. Wrote the paper: MO FO. Contributed to the critical appraisal of the paper and approved the final version: MO KT MS YS FO.
Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer’s disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin), a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.
Chitin, a linear polymer of β-1-4-linked
The Chit1 level is markedly elevated in the plasma of patients with Gaucher disease, an autosomal recessive lysosomal storage disorder
Both Chit1 and AMCase are secreted proteins with molecular weights of approximately 50 kDa. Both proteins contain an N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain
Mammalian chitinases have attracted considerable attention due to their increased expression in individuals with different pathological conditions. Chit1 is increased in individuals with Gaucher disease
The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes are important steps in gaining insight into the
In the present investigation, we developed a quantitative real-time RT-PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. Our results indicate that AMCase is a major transcript in the mouse stomach, suggesting that the corresponding protein functions as a digestive enzyme that breaks down chitin-containing foods and as part of the host defense against chitin-containing pathogens in the gastric contents.
The Mouse Total RNA Master Panel (Clontech Laboratories) was used to examine the tissue distribution of transcripts. We analyzed four different embryonic stages and eight adult tissues. In addition, RNA was isolated from the lungs and stomachs of 3-month-old male mice. C57BL/6J mice (CLEAR Japan) were bred at the RIKEN Brain Science Institute Animal Facility. All animal experiments were performed in compliance with the institutional guidelines. The protocol was approved by the Committee on the Ethics of Animal Experiments of the RIKEN Brain Science Institute (Approval No. H19-2B013). All surgery was performed by using diethyl ether as an anesthetic, and all efforts were made to minimize suffering. Those tissues for mRNA preparation were provided by Drs. Miyazaki and Nukina at RIKEN Brain Science Institute. Total RNA was prepared from lungs and stomachs using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. To remove the trace amounts of contaminating genomic DNA, the total RNA samples were treated with RQ1 RNase-Free DNase (Promega) according to the manufacturer’s recommended protocol. The concentrations of the nucleic acids were determined by measuring the absorbance at 260 nm using a BioPhotometer Plus (Eppendorf). Each of the total RNA samples (3 µg) was subjected to reverse transcription with random hexamers as primers. The reaction mixture (15 µl) contained the enzyme buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl2], 100 ng of random hexamers, 10 mM dithiothreitol, and 0.5 mM deoxynucleotide triphosphates (dNTPs). After heating the solution to 60°C for 5 min and incubating the mixture at 37°C for 5 min, 200 U of recombinant murine leukemia virus reverse transcriptase (Invitrogen) was added, and the mixture was incubated at 37°C for 45 min. The reverse transcription was terminated by heating to 95°C for 5 min.
Primers for real-time RT-PCR were designed based on Primer Express Software (Applied Biosystems) and were synthesized commercially (Sigma-Genosys, Sigma-Aldrich). The PCR reactions were performed in a final volume of 13 µl containing 2 × SYBR Green Master Mix (Brilliant II SYBR Green QPCR Master Mix, Agilent), 2.7 ng of mouse cDNA or appropriate dilutions of the external standards (see below), and the appropriate concentrations of the primers for the chitinases, pepsinogen C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin. Standard real-time PCR conditions for the system (Mx3005P, Agilent) were used: an initial denaturation and polymerase activation step for 10 min at 95°C, followed by 40 cycles of denaturation at 95°C for 1 min, 55°C for 30 sec, and 72°C for 1 min. Melting curves were generated after amplification. The PCR products were electrophoresed on a 10% polyacrylamide gel and analyzed using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare). The PCR solution was treated with ExoSAP-IT (USB Products) according to the manufacturer’s instructions to eliminate unincorporated primers and dNTPs, and the products were sequenced using the ABI PRISM Big-Dye Terminator v3.1 Cycle Sequencing Kit and a 3130 Genetic Analyzer (Applied Biosystems). The nucleotide sequences of the primers selected for the real-time PCR are shown in
The standard template cDNA (913 bases) for the quantification of transcript levels by real-time PCR was constructed as follows. The cDNA fragments covering the PCR-target region plus 9–120 bases of the flanking regions of AMCase, Chit1, pepsinogen C, GAPDH, and β-actin were amplified from mouse stomach cDNA by PCR using KOD Plus DNA polymerase (Toyobo) and oligonucleotides primer containing the restriction sites of
To validate our absolute real-time PCR method, we prepared full coding cDNAs by PCR using the primer sets listed in
The molar concentration of the multigene-containing DNA standard was calculated based on the concentration and the molecular weight. Serial dilutions were prepared starting with the standard template concentration, which yielded a Ct of approximately 13 (Ct = fractional threshold cycle value). The standard DNA was subjected to 10-fold serial dilutions, ranging from 100 to 107 molecules, and the aliquots were kept frozen at −20°C until use.
Each sample was amplified in triplicate, and each experiment was repeated at least two times. Using the standard curve, the numbers of Chit1, AMCase, GAPDH, β-actin, and pepsinogen C mRNA molecules were extrapolated automatically by using the MxPro QPCR Software version 4.10 (Agilent). All values were expressed as molecules per 10 ng of total RNA. In some instances, the values were normalized against the levels of GAPDH or β-actin mRNA.
For evaluating our present methodology, we also employed the ΔΔ Ct method
The establishment of a reliable method for the measurement of the expression of chitinase transcripts is an important step in investigating the significance of mouse chitinases. Real-time RT-PCR is, at present, the most sensitive quantitative method for the detection of both low-abundance mRNAs and abundant transcripts. We first compared the gene expression levels of the Chit1 and AMCase genes (see
The expression levels of the Chit1 and AMCase genes were compared. To evaluate the chitinase levels, we used two housekeeping genes, GAPDH and β-actin, which are constitutively expressed at high levels in most tissues and cells. In addition, we chose pepsinogen C, which is a major component of the gastric mucosa, as a reference gene for the stomach tissues. Using these three reference genes, we evaluated the gene expression levels of Chit1 and AMCase in mouse tissues.
We first designed several sets of primers for quantitative PCR and evaluated their suitability based on whether they gave single products, as reflected by a single melting temperature (Tm) and a single band on a 10% polyacrylamide gel. The nucleotide sequences of the products were also verified. To examine the specificity of the primers, each of the PCR products was amplified under real-time PCR conditions by using a mouse tissue cDNA mixture consisting of tissues from four embryonic stages and eight adult tissues and was assayed in combination with different methods. As shown in
PCR primers were selected based on whether they gave one melting temperature (A–E) and a single PCR product on a 10% polyacrylamide gel (F). To verify the specificity of the primers, the dissociation curves of the PCR products for five genes generated using mouse tissue cDNA mixture were examined. The PCR products were analyzed on a 10% polyacrylamide gel, followed by ethidium bromide staining. The nucleotide sequences of the PCR products were confirmed.
A. Schematic representation of the standard template DNA used for real-time PCR. The target fragments of the AMCase, pepsinogen C, Chit1, GAPDH and β-actin cDNAs with flanking sequences and restriction sites were amplified and ligated in a one-to-one ratio into a DNA fragment, which was then cloned into a cloning vector. The linearized standard template DNA was amplified from the plasmid DNA and used as the standard DNA. B. Nucleotide sequence of the standard template DNA. The template DNA, 913 nucleotides long, contained five cDNA fragments (shown in different colors) covering the PCR-target region (shown in bold underline) plus 9–120 bases of the flanking regions and containing restriction sites for
We sought to compare the expression levels of two chitinases and three reference genes on the same scale (
The quantification of both the chitinases and the reference mRNAs relies on standard curves. Serial dilutions of the standard template DNA were used to construct a standard curve to compare and evaluate the real-time RT-PCR quantification strategies that were used to analyze the five mRNAs. Each standard curve was generated using 10-fold serial dilutions of the standard DNA and the five different primer pairs (
The checked DNAs are as follows: A, Chit1; B, AMCase; C, GAPDH; D, β-actin; E, pepsinogen C. (Left) Real-time PCR quantification of each 10-fold serial dilution of the standard template DNA using primer pairs targeting each gene. (Right) The standard curves by using the standard template DNA containing the five cDNA fragments (brown closed circles). In addition, the quantification of full coding cDNAs was carried out using primer pairs for each gene. The target cDNA was amplified from a dilution of the full coding cDNA with a known concentration (see
To test the absolute equality of the curves, a known concentration of the full coding cDNA was amplified and subsequently analyzed as an unknown sample. This assay was performed to verify that each tested dilution resulted in the expected quantity. As shown in
To study the
Quantification of Chit1 (A) and AMCase (B) mRNAs in mouse tissues. Both chitinases were quantified by real-time PCR using the standard template DNA. All values obtained were expressed as molecules per 10 ng of total RNA. C. Ratios of AMCase to Chit1. All mRNA copy numbers were derived based on the same standard dilutions. The upper panel indicates the actual number, whereas the lower panel shows the logarithm of the values.
The expression levels of the five genes determined using the cDNAs prepared from lung (A) or stomach (B) tissues from 3-month-old mice (n = 5) were quantified by real-time PCR. The upper panel indicates the actual value, whereas the lower panel shows the logarithm of each value.
We then compared the ratios of AMCase to Chit1. The copy number of each mRNA was determined using the same standard dilutions. We found that the stomach and the submaxillary gland predominantly expressed AMCase mRNA (
We next examined the relative quantification in the original data presented in
Many studies have been carried out on the pathophysiology of mammalian chitinases in lung tissues. In this study, we showed that AMCase mRNA is predominantly expressed in mouse stomach tissues (
In stomach tissues, when the Chit1 level was set at 1.0, the relative expression levels were AMCase, 721; pepsinogen C, 2261; GAPDH, 61; and β-actin, 127 (
We also re-examined relative expression of Chit1, AMCase and pepsinogen C shown in
Both Chit1 and AMCase are thought to aid in the host defense against chitin-containing pathogens
Quantitative real-time RT-PCR represents an important advance in quantifying mRNA and allows the detection of the expression of a particular gene of interest at the molecular level. There are many reports on the detection of extremely low levels of mRNA using this technology. However, real-time RT-PCR is not a widely used because it still has limitations in quantifying multiple mRNAs using the same scale. We constructed a standard template DNA comprising five cDNA fragments (each ∼200 bases long). These fragments included fragments for two chitinases, one marker and two housekeeping genes and were combined in one-to-one ratios (
Relative quantification is easier to perform than absolute quantification because a calibration curve is not necessary. In relative quantitation, mRNA levels of the gene of interest are compared from those of the housekeeping genes
In the present investigation, we found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach relative to the levels of housekeeping genes (
Hydrochloric acid is secreted in the stomach, creating acidic conditions for the digestion of proteins by pepsin at approximately pH 2
The highest level of Chit1 was also expressed in the mouse stomach. However, the Chit1 expression level in the stomach was 1/721 that of the AMCase expression level and was lower than the expression levels of two housekeeping genes, GAPDH and β-actin (
Although AMCase is expressed predominantly in the stomach and submaxillary glands, all other tissues examined in this study expressed low, but detectable, levels of Chit1 and AMCase. The expression levels of Chit1 and AMCase were lower than those of the housekeeping genes in these tissues. Chit1 shows a broad pH optimum at around pH 5
More AMCase mRNA than Chit1 mRNA was observed in most mouse tissues, except for the eyes. Our results also suggest that gene expression of Chit1 may be developmentally regulated, suggesting that it plays a role in ontogeny. Studying the regulation of the expression of the mammalian chitinases could give insights into the physiological roles of these enzymes. A detailed characterization of the promoter regions of Chit1 and AMCase genes as well as the identification of
Although mammals do not produce chitin or chitin synthase, they are continually exposed to this polymer through exposure to chitin-containing parasites and pathogens. The substrate for mammalian chitinases is presumably environmental chitin, such as that found in fungi or parasitic nematodes. In the lungs, both enzymes can act as part of the host defense mechanism against chitin-containing pathogens, such as environmental mold and mites, that induce airway allergy. In addition, AMCase in the mouse stomach also plays a specific role in food processing.
Data on the pathophysiological function of AMCase in the human system are contradictory
We established a quantitative real-time PCR system to evaluate the expression levels of two mammalian chitinases, two housekeeping genes and one stomach marker gene using a laboratory-constructed standard DNA. AMCase is predominantly expressed in mouse stomach, where the AMCase mRNA level is comparable to that of pepsinogen C, a major digestive enzyme in the gastric juice. Thus, AMCase is a major transcript in the mouse stomach.
(DOC)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(DOC)
(DOC)
(DOC)
We are grateful to Drs. H. Miyawaki and N. Nukina for providing mouse tissues and encouragements, to Dr. Y. Imamura for valuable suggestions and to Dr. L. L. Isom for critical reading of this manuscript.