We report the identification of a novel secreted peptide, INM02. The mRNA transcript of human INM02 gene is about 3.0 kb. Its open-reading frame contains 762 bps and encodes a protein of 254 amino acids. Northern blot analysis demonstrates that INM02 mRNA is widely expressed in rat tissues, especially with abundant quantities in pancreatic islets, testis, and bladder tissue. We have expressed recombinant INM02 protein and generated rabbit anti-INM02 polyclonal antibodies. We show here that INM02 could be detectable in human serum by ELISA. We also present evidence that INM02 mRNA expression could be regulated by glucose. Experiments on both MIN6 cells and intact isolated islets demonstrate that INM02 mRNA levels are increased more than threefold by high glucose (25 mM) when compared with low glucose (5.5 mM). ELISA analysis shows that secretion of INM02 is significantly augmented by high glucose in vitro. It is speculated that as a novel secreted protein, INM02 is associated with functions of pancreatic islets, especially of β-cells.
The gene content of the mammalian genome is a topic of great importance in the field of molecular biology. Since draft sequences of human genome have been completed (Lander et al. 2001, Venter et al. 2001), genomic sequences of many other mammalians are now available in GenBank and other databases. However, challenges remain to correctly identify all of the encoded genes. To facilitate gene identification efforts and to catalyze experimental investigation, the National Institutes of Health launched the Mammalian Gene Collection program with the aim of providing freely accessible, high-quality sequences for validated, complete open-reading frame (ORF) cDNA clones, and cloned more than 15 000 full-length human and mouse cDNA sequences, which contained complete ORF (Strausberg et al. 2002). Another large-scale effort, termed Secreted Protein Discovery Initiative, utilized multiple gene-identification strategies and identified more than 1000 novel secreted and transmembrane proteins (Clark et al. 2003). In previous studies of our group, cDNA libraries of human hypothalamus, pituitary, adrenal, and insulinoma tissues were established and several novel full-length cDNAs were cloned (Hu et al. 2000, Wang et al. 2004).
Glucose is one of the most important factors by which the expression of genes in either β-cells or other cells of islets are regulated. Many glucose-responsive genes have been identified in β-cells or islets. Webb et al. (2000) identified glucose-responsive expression of many large functional clusters of genes with experiments on MIN6 cells. They also reported that these genes were differentially regulated in high (25 mmol/l (mM)) versus low (5.5 mM) glucose (Webb et al. 2000). Large-scale gene expression analysis of mRNA species from islets of the spontaneously diabetic Goto–Kakizaki (GK) rats and normal control were performed after 48 h of culture at 3 or 20 mM glucose. Specific glucose-regulated genes involved in glucose sensing, phosphorylation, incretin action, glucocorticoid handling, ion transport, mitogenesis, and apoptosis were found disturbed in islets from GK rats when compared with normal controls (Ghanaat-Pour et al. 2007). In another study, differential gene expression profiles were established in isolated intact human islets at low and high glucose, and a highly glucose-regulated transforming growth factor-β signaling cascade was found existing in human islets (Shalev et al. 2002).
In the present study, a novel full-length cDNA named as INM02, which encoded a new secreted protein, was cloned from a cDNA library of human insulinoma tissue. We also presented evidence that both INM02 mRNA and its protein secretion could be regulated by glucose.
Materials and Methods
Sprague–Dawley (SD) rats obtained from the Experimental Animal Center, Shanghai Medical College of Fudan University were used for these studies. Rats were on a 12 h light:12 h darkness cycle at room temperature of 22±1 °C and received food and water ad libitum. All experimental procedures were approved by the Shanghai Animal Care and Use Committee on Animals and followed the policies issued by the International Association for the Study of Pain on the use of laboratory animals. All efforts were made to minimize animal suffering and reduce the number of the animals used.
Cloning and bioinformatics analysis of the full-length cDNA of INM02
A cDNA library of human insulinoma was screened and a novel expressed sequence tag (EST) was selected. A BLAST search on the novel EST was performed in GenBank and several homologous ESTs were obtained. Assemblies were carried out between the novel EST and its homologous ESTs by using Autoassembler software (Applied Biosystem, Foster City, CA, USA). Overlapping EST sequences were assembled into contigs. The contigs were checked to see whether they contained ORF by using DNA Strider 1.2 software (Applied Biosystem). The 5′ or 3′ sequences of the contigs were selected to perform a BLAST search for homologous ESTs in dbEST and other databases, and then assemblies were repeated until the complete ORF of a novel gene was finished. A BLAST search of the nucleic acid sequences and translated amino acid sequences of the novel gene were performed in GenBank and protein database or other databases. There was little homolog to known genes and proteins. Nucleic acid sequences of the novel gene were amplified by RT-PCRs with the following primers: forward primer: 5′-TTCGCTGGTGGGAAGAAGCCGAGATG, reverse primer: 5′-TGAGGAACAGGACGACGGGAATGATG. The PCR conditions (Advantage-GC2 PCR kit (Clontech)) were 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, and 68 °C for 2 min. The PCR product was recovered and purified by QIAquick gel extraction kit (Qiagen), and then sequenced by ABI 3770 sequencer (Applied Biosystem). After the molecular cloning of INM02 was finished, several online softwares were used to predict functional domains in INM02. For example, software SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) was used to predict signal peptide in amino acid sequences of INM02 protein.
Northern blot analysis
Northern blotting was performed using nonisotopic digoxigenin (DIG) northern starter kit (Roche Diagnostics) according to the manufacturer's protocol. Target gene fragments, rat INM02, and β-actin were cloned into pDrive vector (Qiagen) and confirmed by restriction enzyme digestion and sequencing analysis. DIG-labeled probes were generated by transcription with SP6/T7 RNA polymerase using the DIG RNA labeling kit (Roche Diagnostics). Total RNA was isolated by Trizol reagent (Invitrogen). Adequate amounts of RNA were electrophoresed on a 1.2% agarose-formaldehyde denaturing gel and were transferred by capillary blotting to positively charged nylon membranes (Roche) in 20×SSC. The membrane was then baked at 120 °C for 30 min. Hybridization was performed at 68 °C with agitation overnight. The membrane was washed twice with 2×SSC and 0.1% SDS for 5 min at room temperature, and twice with 0.1×SSC and 0.1% SDS for 15 min at 68 °C. After stringency washes, the membrane was blocked by blocking solution (Roche) and then incubated with anti-DIG serum/alkaline phosphatase (AP) conjugate (Roche) for 30 min respectively. CDP-Star (Roche) was used as the chemiluminescence substrate. Signals were visualized on X-ray film. The intensity of any RNA signal was quantified by densitometric scanning and statistical differences of Inm02 mRNA levels were evaluated using one-way ANOVA.
The truncated ORF sequences of human INM02 gene excluding the nucleotide sequences by which the predicted signal peptide was deduced and amplified using the following primers: 5′-ACCTGAATTCAGCGGCTGCCGGGCCGGGACTGG and 5′-TAGTAAGCTTAGGCCTCCTGTGGCGGTGGCGCGG (sequences of EcoRI and HindIII are underlined). The PCR fragment was subsequently subcloned into pPROEX HTb vector (Invitrogen). The construct of pPROEX HTb-INM02 was verified by sequencing analysis.
Expression and purification of His-INM02 fusion protein
The recombinant pPROEX HTb-INM02 vector was transformed into Escherichia coli strain DH5α. Positive clones by ampicillin screening were cultured in 5 ml LB medium with ampicillin (50 μg/ml), shaking at 250 r.p.m. overnight, and then an aliquot of 3 ml culture was incubated to 300 ml new culture medium to the optical density of 0.6 (OD 600 nm). The fusion protein expression was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 3 h at 25 °C. The cells were then harvested by centrifuging at 6000 g for 15 min at 4 °C. The cell pellet was resuspended in 1×PBS (10 mM Na2HPO4, 1.75 mM KH2PO4, 13.7 mM NaCl, and 2.65 mM KCl, pH 7.4 at 25 °C) and sonicated on ice to ensure full lysis. The lysate was centrifuged at 20 000 g for 30 min at 4 °C, and then the supernatant was purified by using nitrilotriacetic acid affinity (Ni-NTA) columns (Invitrogen) according to the manufacturer's instruction. The purified fractions were analyzed by 12% SDS-PAGE, followed by Coomassie brilliant blue R250 staining. The protein density was measured by using Coomassie protein assay (Pierce, Rockford, IL, USA) reagent on Biophotometer (Eppendorf, Hamburg, Germany).
Generation of rabbit polyclonal antiserum
Purified recombinant INM02 (500 μg) was emulsified in complete Freund's adjuvant and injected subcutaneously into a 6-month-old male rabbit. Additional immunizations with 200 μg were administered on days 44 and 70 in incomplete Freund's adjuvant. Serum was collected 1 month after the third immunization, and then antibody titers against the recombinant protein was tested by ELISA.
Western blot analysis
One hundred nanograms of recombinant INM02 protein was loaded onto a gel, subjected to SDS-PAGE (5% stacking gel, 12% running gel) and then electroblotted onto nitrocellulose membranes. Membranes were blocked in Tris-buffered saline (TBS) buffer, pH 7.6 containing 5% skimmed milk powder, at 4 °C overnight, then exposed to anti-INM02 polyclonal antibodies (at a dilution of 1:4000) in TBS-Tween buffer containing 5% skimmed milk powder for 2 h. Membranes were then washed and incubated with anti-rabbit IgG (DAKO, Carpinteria, CA, USA) conjugated to HRP diluted 1/1000 in the same buffer for 1 h. After a series of washes in TBS-Tween buffer, protein bands were visualized by chemiluminescence with an ECL luminescence kit (Pierce) and exposed to X-ray film.
Establishment of sandwich ELISA to detect INM02
First, polyclonal IgG antibodies were purified from the rabbit antiserum by using protein A agarose (Amersham Biosiences) and were then labeled with HRP (Pierce) according to the manufacturer's instruction. Aliquots of the purified antibodies (1 μg/well) were incubated for 16 h at 4 °C in flat-bottomed 96-well plates (GenScript, Piscataway, NJ, USA). After three washes with PBS, 100 μl of serum samples in which INM02 would be detected were added and incubated for 2 h at room temperature under constant shaking. Serial dilutions of human recombinant INM02 were included as standards in each experiment. The plate was washed three times with PBS containing 0.1% Tween 20 in an ELISA wash station (Thermo Scientific, Waltham, MA, USA). HRP conjugated antibodies (dilution 1:500) were then incubated for 1.5 h under constant shaking at room temperature. Wells were washed four times with PBS containing 0.1% Tween 20 and developed using the TMB two-component peroxidase substrate solution (Kirkegaard & Perry, Gaithersburg, MA, USA). The reaction was stopped by adding 1 M H3PO4 and quantitated by absorbance at 450 nm using a Thermo ELISA reader (Thermo Scientific).
To determine the cellular distribution of INM02 in pancreatic tissue, double immunohistochemical staining was performed. Briefly, paraffin-embedded sections (5 μm) of rat pancreatic tissues were deparaffined and rehydrated. After antigen retrieval, the sections were treated with 3% cold hydrogen peroxide to suppress endogenous peroxidase activity and then blocked with 0.2% Tween 20/3% BSA/TBS. Rabbit anti-INM02 polyclonal antibody (1:200), which was generated in our lab and goat anti-insulin monoclonal antibody (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), was used to incubate the same section sequentially. After three washes with PBS, the sections were incubated with anti-rabbit IgG antibody conjugated with AP (1:300) (Pierce), and then with 5-bromo-4-chloro-3′-indolylphosphate p-toluidine/nitro-blue tetrazolium chloride (Kirkegaard & Perry) to stain INM02. The sections were then observed and imaged by using a Leica microscope (Leica Microsystems, Watzlar, Germany). The sections were washed with PBS and then incubated with HRP-conjugated anti-goat IgG antibody (1:300) (Pierce) followed by being applied with AEC substrate buffer (Santa Cruz Biotechnology) to stain insulin. The sections were imaged again to show distributions of INM02 and insulin in pancreatic tissues.
MIN6 cells (passage 22–26) were cultured in DMEM (Invitrogen) with 25 mM glucose, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 μl/l β-mercaptoethanol at 37 °C and 5% CO2. Cells were seeded at 1×106 per well in 6-well plates and then cultured in DMEM containing different glucose concentrations (5.5 and 25 mM) for different periods (1, 24, and 48 h) respectively. INM02 mRNA levels were quantitated by using northern blotting described as above.
Islet isolation and treatment
Islets of Langerhans were isolated from male SD rats by in situ pancreas collagenase infusion and separated by density gradient centrifugation according to the protocol described by Kinasiewicz et al. (2004). The concentration of collagenase type XI was 0.5 mg/ml. Freshly isolated rat islets were cultured overnight in DMEM containing 5.5 mM glucose, 10 mM HEPES, 0.5% BSA, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2. To determine whether INM02 mRNA expression was regulated by glucose, isolated islets were transferred to 24-well plates (10 islets/well) and cultured in DMEM containing different glucose concentrations (5.5 and 25 mM) for different periods (1, 24, and 48 h) respectively. INM02 mRNA levels were quantitated by using QuantiFast SYBR Green PCR kit (Qiagen) according to the manufacturer's instruction. Statistical differences were evaluated using one-way ANOVA.
Cloning and bioinformatics analysis of INM02 gene
Human INM02 gene was mapped on chromosome 19q13.3–4 stretching 16.5 kb. The mRNA transcript of INM02 was about 3.0 kb. INM02 gene had 12 exons and its ORF contained 762 bp, which encoded a protein of 254 amino acids. Figure 1A demonstrated the nucleotide and amino acid sequences of human INM02, and the sketch of genomic structure of INM02 gene. The sequence data of INM02 gene had been submitted to GenBank databases under accession number
Online software-based prediction analysis showed that there was a typical signal peptide locating at amino terminal of INM02 protein (as shown in Fig. 2C). Otherwise, no functionally conserved domains were predicted. After a BLAST search of the nucleotide sequence of human INM02 gene was performed in Ensembl database (
Expression of INM02 mRNA and protein in rat tissues
To study the tissue distribution of INM02 mRNA, SD rats were selected as the animal models and the rat homologous gene was cloned. The expression profile of INM02 was investigated in multiple tissues of rats by using northern blotting. The result of northern blotting demonstrated that Inm02 mRNA was widely expressed in rat tissues, especially with abundant quantities in pancreatic islets, testis, and bladder tissue (as shown in Fig. 3A). Since Inm02 mRNA was highly expressed in pancreatic islets, it was necessary to investigate the cellular localization of INM02 in islets or pancreatic tissue. Immunohistochemical staining was performed to detect INM02. Meanwhile, staining of insulin was used as a control to distinguish islets from exocrine tissue of pancreas. Immunohistochemical analysis demonstrated that INM02 was expressed in islets of Langerhans, and it was hardly detected in exocrine tissue of pancreas (as shown in Fig. 3B).
Purification of recombinant INM02 protein and generation of anti-INM02 polyclonal antibody
After transfection of recombinant pPROEX HTb-INM02 vector into E. coli and IPTG induction, a prominent band of ∼32 kDa, which was agreed with the predicted size of recombinant protein, was detected by SDS-PAGE. The fusion protein was purified from E. coli lysates by Ni-NTA chromatography (as shown in Fig. 2A).
After three immunizations of a rabbit with recombinant INM02 protein, the rabbit serum containing anti-INM02 polyclonal antibody was collected, and then high titer antibody activity was tested by using ELISA. One hundred nanograms of recombinant INM02 protein was loaded onto a 12% SDS-PAGE gel and detected with the rabbit antiserum as primary antibody. The result of western blotting showed that the polyclonal antibody against INM02 was specific and validated (as shown in Fig. 2B).
Detection of INM02 in human serum by ELISA
We developed a sandwich ELISA to investigate whether INM02 was detectable or not in human serum. Human recombinant INM02 with serial dilutions were used as standards in the experiment. The corresponding standard curve exhibited a high linearity. Totally, 50 people including 30 patients hospitalized in our hospital and 20 healthy controls were recruited and their serum was collected. INM02 was detected in their serum by using developed ELISA. We set the detectable threshold of two times more than OD at 450 nm of negative control. The ELISA analysis demonstrated that INM02 was detectable in human serum (as shown in Fig. 2D). Additionally, we investigated the difference in serum INM02 between diabetic patients and non-diabetic controls. Totally, 18 new-diagnosed diabetic patients (according to the criteria for diagnosis of diabetes recommended by the American Diabetes Association in 1997) and 19 non-diabetic controls were recruited in our study and their serum INM02 levels were investigated. ELISA analysis demonstrated that serum INM02 of the diabetics were lower than that of the controls. Statistical analysis showed, however, that there was no significant difference between the two groups (P value: 0.157).
Regulation of INM02 mRNA expression and its protein secretion by glucose
To determine whether INM02 mRNA expression or its protein secretion was regulated by glucose, MIN6 cells were cultured at low (5.5 mM) and high glucose (25 mM) for various periods (1, 24, and 48 h) respectively. As shown in Fig. 4A–C, neither INM02 mRNA expression nor its protein secretion had significant differences between low and high glucose for 1 h culture time. Compared with 5.5 mM glucose, however, 25 mM glucose increased INM02 mRNA levels 3.5-fold (P<0.001) and 3.4-fold (P<0.001) for 24 and 48 h culture time respectively. Meanwhile, secretion of INM02 protein were augmented 2.1- (P<0.01) and 3.2-fold (P<0.01) at high glucose for 24 and 48 h culture time respectively when compared with low glucose.
We next determined whether changes in glucose concentration and culture time might affect expression of INM02 mRNA or secretion of its protein in intact isolated rat islets. Similar results with experiments on MIN6 cells were obtained in isolated islets. Culture of isolated islets for 1 h at high glucose didn't significantly affect expression of INM02 mRNA or secretion of its protein when compared with low glucose. With the culture time prolonging, high glucose significantly increased both INM02 mRNA levels and its protein secretion. There were 3.8- (P<0.001) and 3.9-fold (P<0.001) augmentations of INM02 mRNA levels at 25 mM glucose for 24 and 48 h culture time respectively when compared with 5.5 mM glucose. Compared with low glucose, high glucose significantly increased secretion of INM02 protein during 24 h (2.3-fold versus 1 h, P<0.01) and 48 h (3.1-fold versus 1 h, P<0.01) culture periods (Fig. 4E and F).
Meanwhile, we also performed experiments to investigate insulin secretion regulated by glucose in MIN6 cells and islets. The results of RIA indicated that high glucose significantly increased the secretion of insulin in both MIN6 cells and islets during 1, 24, and 48 h culture periods (as shown in Fig. 4D and G).
In our previous studies, a cDNA library of human insulinoma tissue was constructed and gene expression profile of insulinoma was established. Several novel genes were cloned from the cDNA library, and their nucleic acid and amino acid sequences were submitted into GenBank (Wang et al. 2004). Among the novel genes, INM02 was selected for extensive study.
Signal peptides control the entry of virtually all proteins to the secretory pathway, both in eukaryotes and prokaryotes. They comprise the N-terminal part of the amino acid chain and are cleaved off while the protein is translocated through the membrane (von Heijne 1990, Rapoport 1992). Computational methods for prediction of N-terminal signal peptides were published about 20 years ago, initially using a weight matrix approach (McGeoch 1985, von Heijne 1986). SignalP, one of the currently most used methods, predicts the presence of signal peptides in proteins. SignalP 3.0, the newest version of SignalP, was presented by Bendtsen and showed in all cases good performance over prediction of signal peptides (Bendtsen et al. 2004). SinalP 3.0 analysis showed that a typical signal peptide consisting of 27 amino acids existed in the N terminal of INM02 protein. The predicted cleavage site was located between glycine and serine. We generated anti-INM02 polyclonal antibody, which consisted of different antibodies against various antigenic determinants of INM02. A sandwich ELISA to detect INM02 was established by using anti-INM02 polyclonal antibody as the capture antibody, and HRP conjugated anti-INM02 antibody as the detection antibody. We detected 50 human serum samples by the developed ELISA and found INM02 protein existing in all the samples in different amounts. INM02 was also detectable in mediums culturing MIN6 cells and isolated islets. Furthermore, it was shown that secretion of INM02 protein was regulated by glucose. Based on the above analysis, it was confirmed that INM02 was a secreted protein.
We presented evidence that both INM02 mRNA and protein were highly expressed in islets of Langerhans, which constituted the endocrine portion of the pancreas. Glucose is one of the most important metabolic factors, which regulate the function of islets or β-cells. In order to investigate whether INM02 mRNA level or its protein secretion was regulated by glucose, we treated MIN6 cells and intact rat islets with different concentrations of glucose for various culture times. In vitro study on isolated intact islets could mimic the physiological condition of islets in vivo. β-cells account for 75% population of mouse islet cells. MIN6 cells provide a homogeneous β-cell population that responds synchronously and physiologically to changes in glucose concentration (Miyazaki et al. 1990, Skelly et al. 1996). Similar results were observed in experiments with MIN6 cells and intact rat islets. Both expression of INM02 mRNA and secretion of its protein could be regulated by glucose in vitro. In detail, high glucose increased expression of INM02 mRNA more than threefold when compared with low glucose. Meanwhile, secretion of INM02 protein was significantly augmented by high glucose. It is well known that insulin secretion of β-cells is tightly regulated by glucose. In our study, compared with low glucose, high glucose significantly increased the secretion of insulin in both MIN6 cells and islets during 1, 24, and 48 h culture periods. The above data indicated that the pattern of INM02 secretion regulated by glucose was similar to that of insulin. In order to exclude the effects of insulin on expression of INM02 gene, MIN6 cells were cultured at 10, 50, and 100 nM insulin for 24 h. Northern blot data indicated that there were no significant differences of INM02 mRNA levels between various concentrations of insulin (data not shown). ELISA analysis demonstrated that serum INM02 of the diabetics were lower than that of the controls. But there was no significant difference between the two groups. According to our present results of experiments on INM02, it is certain that high glucose increased INM02 mRNA levels and its protein secretion in islets or β-cells. It is, however, uncertain that INM02 is related to diabetes. It is also very difficult to assess the importance of INM02 for diabetes. It is well established that dysfunction of β-cells plays an important role in the pathogenesis of diabetes, and several glucose-responsive genes are involved in the process. In the study, we have presented evidence that INM02 is a glucose-responsive gene, and its protein secretion is also regulated by glucose. The relationship between INM02 and function of β-cells or diabetes is worthwhile to be deeply investigated in the future.
In conclusion, we have successfully cloned the full-length cDNA of human INM02 gene and investigated its tissue distribution. We also have expressed recombinant INM02 protein and generated rabbit anti-INM02 polyclonal antibody. We presented evidence that INM02 was a secreted protein, which could be detectable in human serum. We also presented evidence that both INM02 mRNA expression and its protein secretion were significantly increased by high glucose. It was speculated that as a novel secreted protein, INM02 was associated with functions of pancreatic islets, especially of β-cells.
Declaration of interest
We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This work was supported by grants to R H from the Chinese High Tech Program (2002BA711A05), the National Natural Science Foundation of China (30670999, 30770854 and 30711120573) and the Shanghai Science and Technology Commission (08DJ1400605, 08JC1403200). The National Natural Science Foundation of China also provided grants to X W (30300162) and Z Y (30871197).
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