Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues.

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific

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

Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N\u27-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N\u27-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases

Heterogeneous Tumor-Immune Microenvironments between Primary and Metastatic Tumors in a Patient with ALK Rearrangement-Positive Large Cell Neuroendocrine Carcinoma

Evolution of tumor-immune microenviroments (TIMEs) occurs during tumor growth and dissemination. Understanding inter-site tumor-immune heterogeneity is essential to harness the immune system for cancer therapy. While the development of immunotherapy against lung cancer with driver mutations and neuroendocrine tumors is ongoing, little is known about the TIME of large cell neuroendocrine carcinoma (LCNEC) or anaplastic lymphoma kinase (ALK) rearrangement-positive lung cancer. We present a case study of a 32-year-old female patient with ALK-rearrangement-positive LCNEC, who had multiple distant metastases including mediastinal lymph-node, bilateral breasts, multiple bones, liver and brain. Multiple biopsy samples obtained from primary lung and three metastatic tumors were analyzed by fluorescent multiplex immunohistochemistry. Tissue localizations of tumor-infiltrating lymphocytes in the tumor nest and surrounding stroma were evaluated. T cell and B cell infiltrations were decreased with distance from primary lung lesion. Although each tumor displayed a unique TIME, all tumors exhibited concomitant regression after treatment with an ALK-inhibitor. This study provides the first evidence of the coexistence of distinct TIME within a single individual with ALK-rearrangement-positive LCNEC. The present study contributes to our understanding of heterogeneous TIMEs between primary and metastatic lesions and provides new insights into the complex interplay between host-immunity and cancer cells in primary and metastatic lesions

Analysis of Chit1 and AMCase mRNAs and reference gene mRNAs in lung and stomach tissues.

<p>The expression levels of the five genes, which were determined using cDNAs prepared from normal human lung (A) and stomach (B) tissues, were quantified by real-time PCR. The upper panel indicates the actual value, and the lower panel shows the logarithm of each value. The expression level of the human Chit1 gene was set to 1.0; the values on the bars indicate the relative expression levels compared to the expression level of the human Chit1 gene.</p

Expression of Chit1 and AMCase mRNAs in human and mouse tissues.

<p>The expression levels of Chit1 (A) and AMCase (B) mRNAs in eight human and mouse tissues were quantified on the same scale by real-time PCR using the hybrid standard DNA. Filled bars, human tissues; hatched bars, mouse tissues. All of the values are expressed as number of molecules per 10 ng of total RNA in y axis. The upper panel indicates the actual value, and the lower panel shows the logarithm of each value. Part of mouse data has been reported in our previous paper <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067399#pone.0067399-Ohno1" target="_blank">[17]</a>.</p

Expression of Chit1 and AMCase mRNAs in normal human tissues.

<p>Quantification of Chit1 (A) and AMCase (B) mRNAs in human tissues. Both chitinases were quantified by real-time PCR using the human standard DNA. All of the values are expressed as number of molecules per 10 ng of total RNA in y axis.</p

Strategy for comparing Chit1 and AMCase mRNA levels between human and mouse tissues.

<p>(A) The expression levels of the Chit1 and AMCase genes in human and mouse tissues were compared. (B) Schematic representation of the human-mouse hybrid standard DNA used for the quantification. The human and mouse standard DNAs were ligated using the <i>Eco</i>RI site at a one-to-one ratio into a DNA fragment and used as the human-mouse hybrid standard DNA.</p

Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of <i>Escherichia coli</i> Possesses Chitinase Functions Comparable to CHO-Expressed Protein

<div><p>Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an <i>E. coli</i>-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in <i>E. coli</i>. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the <i>Staphylococcus</i> Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in <i>E. coli</i>. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl <i>N,N</i>′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl <i>N,N</i>′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly <i>N,N′</i>-diacetylchitobiose fragments. Thus, the <i>E. coli</i>-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.</p></div

The levels of chitinases activity and protein expression in mouse and human stomach tissues.

<p>(A) The chitinolytic activity in the stomach extracts from mouse and human stomach tissues. Filled bars, human tissues; hatched bars, mouse tissues. (left) AMCase activity was determined using the synthetic chitin substrate of 4MU-chitobioside. Chitinolytic activity at pH 2.0 and pH 5.0 were expressed in y axis. (right) Chit1 activity was measured using 4MU-chitotriose at pH 5.2, as previously described in Materials and Methods. AMCase activity was also measured at pH 2.0. All graph points are the mean of triplicate measurements and representative of multiple experiments. (B) Representative patterns of Western blotting of mouse and human AMCase in mouse or human stomach proteins. Proteins were run on SDS-polyacrylamide gels and analyzed by Western blotting using anti-human or anti-mouse AMCase antibodies. Stomach soluble proteins (6.3 µg) were separated on SDS-polyacrylamide gels and transferred onto Immobilon membranes, and probed with AMCase specific antibodies.</p