MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 33, Number 4, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2014.0001

Production and Characterization of a Monoclonal Antibody Against GRAM Domain-Containing Protein 1A Xiuli Song,1 Shihao Wang,1 Bin Gu,1 Qiannv Hou,2 Yajuan Liu,3 and Ming Zhang1

From the proteomic analysis, we identified hundreds of novel proteins that have never been characterized for their expression profile and function on human embryonic stem (hES) cells. In this study, we produced a group of monoclonal antibodies against the GRAM domain-containing protein 1A, which was found on hES cells. Using these antibodies, we analyzed the expression of GRAMD1A in various tissues and tumor cell lines. The results showed that GRAMD1A is expressed in the nucleus and cytoplasm of hES cells, cancer cell lines and ectoderm, mesoderm, and endoderm tissues. The development of the monoclonal antibody to GRAMD1A and the characterization of the expression pattern of this protein could have significant implications in the functional characterization of this novel protein.

Introduction

T

he Human Genome Project (HGP) produced an accurate reference sequence for each human chromosome, with only a small number of gaps, and excluding large heterochromatic regions.(1) The human genome sequence initiated the comprehensive discovery of most human genes, and by inference most human proteins.(2,3) The 1000 Genomes Project’s latest effort mapped more than 38 million singlenucleotide polymorphisms (SNPs), 58% of which were previously unknown, in 14 different worldwide populations.(4,5) The HGP also led to the emergence of proteomics, a discipline focused on identifying and quantifying the proteins present in discrete biological compartments.(6) At present, there is still limited knowledge about the proteins of approximately two-thirds of the 20,300 protein coding human genes mapped through the HGP.(7) Based on the UniProtKB/ Swiss-Prot database content, about 6000 (30%) of these genes currently lack any experimental evidence at the protein level; for many others, there is very little information related to protein abundance, distribution, subcellular localization, interactions, or cellular functions.(8,9) So it is clear that much work remains to be done to identify and uncover the poorly known proteins of humans. Embryonic stem (ES) cells are widely used in basic research and hold great potential for regenerative medicine.(10) Previous studies showed that the gene expression profile of ES cells is highly complicated and promiscuous at mRNA level. Moreover, our proteomics data have shown that both 1 2 3

mouse ES (mES) and human ES (hES) cells express a large variety of functional and tissue specific proteins in a heterogeneous manner. These results indicated that the selfrenewal and differentiation of hES cells are controlled by a very complicated protein network that is not yet dissected. Also from the proteomic analysis, we identified hundreds of novel proteins that have never been characterized for their expression profile and function on hES cells.(11,12) As hES cells represent a developmentally native state of human cells and is highly relevant to regenerative medicine and cancer biology, it would be of value to characterize the expression profile and function of these new proteins in different human cell types and tissues.(13,14) The antibody-based approach is an efficient method for new protein identification and expression profiling. In this study, we produced a group of monoclonal antibodies against the GRAM domain-containing protein 1A, a novel protein we identified on hES cells. Eight mouse monoclonal antibodies were acquired of which the recognition specificity were characterized by ELISA and Western blotting, and one of these antibodies was applicable for immunostaining. Using these antibodies, we analyzed the expression of GRAMD1A in various tissues and tumor cell lines. The results showed that GRAMD1A is expressed in the nucleus and cytoplasm of hES cells, several cancer cell lines and ectoderm, mesoderm, and endoderm tissues. The development of the monoclonal antibody to GRAMD1A and the characterization of the expression pattern of this protein could have significant implications in the functional characterization of this novel protein.

Institute of Genetics, College of Life Sciences, Zhejiang University, Hangzhou, China. HangZhou HuaAn Biotechnology Company, Hangzhou, China. College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, China.

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MONOCLONAL ANTIBODY AGAINST GRAMD1A Materials and Methods Cell culture

Mouse embryonic fibroblast (MEF) feeder cells were isolated from the embryos of BALB/c mice at gestational day 13.5. MEFs were routinely cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37C in 5% CO2 atmosphere. MEFs were inactivated by Mitomycin C and plated at a density of 4 · 104 cells/cm2 for ES culture. Human embryonic stem cell line H9 (hES) were provided by Wicell Research Institute and cultured on Mitomycin C inactivated MEFs in Knockout DMEM (Gibco) supplemented with 20% Knockout SR (Gibco) and 10 ng/mL bFGF (Millipore, Billerica, MA) at 37C in a 5% CO2 atmosphere. Human A431 cells were cultured with F-12 medium (Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich, Saint Louis, MO) and antibiotics (70 mg/mL penicillin and 100 mg/mL streptomycin) at 37C in 5% CO2 atmosphere. Human lung adenocarcinoma A549 cells and human liver carcinoma HepG2 cells were cultured with RPMI 1640 medium (Gibco) supplemented with 10% (v/v) heat-inactivated newborn calf serum (Sigma-Aldrich) and antibiotics (70 mg /mL penicillin and 100 mg/mL streptomycin) at 37C in 5% CO2 atmosphere. Human breast carcinoma SKBR-3 cells, human colorectal cancer cell line SW480,

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HCT116, and human glioma cells SHG-44 were cultured with DMEM medium (Gibco) supplemented with 10% (v/v) heatinactivated fetal bovine serum (Sigma-Aldrich) and antibiotics (70 mg/mL penicillin and 100 mg/mL streptomycin) at 37C in 5% CO2 atmosphere. Human breast cancer cell line MCF-7 were cultured with DMEM medium (Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 0.12 mM NEAA, 1 mM sodium pyruvate, 0.2 U/mL bovine insulin, and antibiotics (70 mg /mL penicillin and 100 mg/mL streptomycin) at 37C in 5% CO2 atmosphere. Mouse fibroblasts NIH/3T3 were cultured with DMEM medium (Gibco) supplemented with 10% (v/v) heat-inactivated newborn calf serum (SigmaAldrich), 4.5 g/L glucose, 4 mM L-glutamine, and antibiotics (70 mg/mL penicillin and 100 mg/mL streptomycin) at 37C in 5% CO2 atmosphere. Bioinformatics

The composition and position of GRAMD1A gene were analyzed by the human genome database at UCSC Genome Bioinformatics site (http://genome.ucsc.edu). The protein sequence was analyzed at European Bioinformatics site (www.ebi.ac.uk/interpro/protein). The position of hydrophobicity region and antigenicity region of it were analyzed by Protean software. The protein 3D structure of GRAM domain-containing protein 1A was predicted using the RaptorX web server.

FIG. 1. Sequence analysis of GRAM domain-containing protein 1A. (A) A gram domain between 145 and 686 amino acids was predicted (www.ebi.ac.uk/interpro/protein/Q96CP6). (B) Sequence analysis of antigenicity and hydrophobicity region using Protean software. (C) 3D structure was predicted using the RaptorX web server (http://raptorx.uchicago.edu/ StructurePrediction/myjobs/9267699_46659).

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side (IPTG). The cells were harvested and lysed in lysis buffer (20 mM Tris, 10 mM NaCl, 1 mM EDTA [pH 7.4]) and loaded onto glutathione sepharose columns (GE Healthcare, Uppsala, Sweden). Columns were washed with washing buffer (20 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA [pH 8.0]) and bound proteins were eluted using elution buffer (20 mM Tris-HCl [pH 8.0], 10 mM GSH). The purified protein was analyzed for its purity by SDS-PAGE. Generation of anti-GRAMD1A MAbs

FIG. 2. Expression of GRAMD1A protein by induction with IPTG at 28C for 4 h in BL21 cells and purification of GRAMD1A antigen. Samples were confirmed by Coomassie blue staining. Lane M, protein molecular weight marker; lane 1, total cell lysate without induction; lane 2, total cell lysate with 0.5 mM IPTG induction; lane 3, total cell lysate with 1 mM IPTG induction; lane 4, eluted antigen.

Five-week-old BALB/c mice were immunized subcutaneously with 100 mg of purified GRAMD1A protein emulsified in Freund’s complete adjuvant (Sigma-Aldrich). Booster injections were carried out thrice at intervals of 2 weeks, with 50 mg of recombinant GRAMD1A protein emulsified in Freund’s incomplete adjuvant. The serum titer was tested with indirect ELISA. Three days before fusion, the mice were injected intraperitoneally with 100 mg of GRAMD1A protein. Spleen cells were isolated and fused with mouse myeloma sp2/0 cells by standard hybridoma fusion techniques and selected by HAT medium. The screening of hybridomas secreting anti-GRAMD1A MAbs was done by indirect ELISA. Positive clones were expanded and subcloned to monoclonality by a standard limiting dilution protocol. The isotypes of the MAbs were determined using a mouse MAb isotyping kit (Pierce, Rockford, IL). Enzyme-linked immunosorbent assay

Recombinant GRAMD1A expression and purification

A fragment encoding the last 94 amino at c-terminal of the GRAMD1A gene was amplified from hES H9cDNA and cloned into pGEX-4T-1 plasmid using a set of primers: 5¢GCGGATCCTACCGCCTCTGGTCCCTG-3¢ with BamHI restriction site; rev: 5¢-GCGAATTCTCAGGAAAAGCTG TCATCGGG-3¢ with EcoRI restriction site. The plasmid was transformed into Escherichia coli strain BL21 and induced using 0.5 mM and 1 mM isopropyl-b-D-thio-galactopyrano-

The 96-well flat-bottomed microtiter plates were coated with recombinant GRAMD1A protein (0.1 mg/well) and incubated overnight at 4C. The plates were washed and then blocked with PBST containing 5% BSA for 1 h at 37C. Hybridoma culture supernatants were added and incubated for 1 h at 37C. HRP-conjugated goat anti-mouse antibodies (Huaan Bio, Hangzhou, China) were added and incubation was continued for 60 min at 37C. Tetramethylbenzidine (TMB)/H2O2 solution was used as a substrate for the enzyme. After 5 min

FIG. 3. ELISA analysis of mouse sera against immunogen GST-GRAMD1A and fusion protein GST. Immune sera were diluted as indicated and incubated with GST-GRAMD1A (violet bar) or GST (red bar). The absorbance (optical density) was measured at 450 nm.

MONOCLONAL ANTIBODY AGAINST GRAMD1A

reaction at 37C, the equal volume of stopping solution (2 M H2SO4) was added. Absorbance was read at 450 nm with a multi-well plate reader (Bio-Rad, Hercules, CA). Immunoblot analysis

The purified recombinant protein GRAMD1A and cell lysates were subjected to SDS-PAGE and transferred onto a polyvinyl difluoride (PVDF) membrane (Millipore) using a trans blot apparatus (Bio-Rad). The membrane was then incubated with 1% BSA at 37C for 1 h followed by incubation with the culture supernatant of hybridomas at 4C overnight. After extensive washing with PBST, the membrane was incubated for 1 h at 37C with HRP-conjugated goat antimouse antibodies (Huaan Bio). The ECL chemiluminescence system was used for detection of the protein bands.

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and has low hydrophobicity residues (Fig. 1B). The protein 3D structure predicted by the RaptorX web server also revealed that the C-terminal domain has fewer alpha (a) helices and beta (b) pleated sheets, which is not preferable in antigen design, compared to the N-terminal and internal domains (Fig. 1C). Based on these analyses, we chose the C-terminal peptides as the region to produce recombinant antigen. Expression of recombinant GRAM domain-containing protein 1A

A full-length cDNA of human GRAMD1A encoding 2672 bp was recruited from hES cell line H9 by RT-PCR. A C-terminal fragment encoding the last 94 amino acids was amplified using the designed primers. The selected region was cloned into vector pGEX-4T-1 with GST-tag. A 282-bp DNA fragment was confirmed by sequencing.

Immunofluorescent staining

The tested cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with PBS containing 0.3% Triton X-100 for 10 min at room temperature. The cells were then incubated with 1% BSA for 30 min at 37C to minimize unspecific binding of the antibodies. The cells were incubated in the diluted MAb GRAMD1A and commercial MAb SSEA4 (R&D, Minneapolis, MN) in a humidified chamber overnight at 4C. Next, the cells were incubated with Alexa555-conjugated goat anti-mouse secondary antibody (Invitrogen) for 45 min at 37C. After washing with PBS, the cell coverslips were mounted with mounting media (Vector Labs, Burlingame, CA) with the counterstain DAPI (4¢,6-diamidino-2-phenylindole) and visualized under a laser-scanning microscope (Carl Zeiss AG, Oberkochen, Germany). Immunohistochemical staining

Paraffin-embedded mouse and human tissue sections were deparaffinized, rehydrated, and incubated with anti-GRAMD1A MAb at 4C overnight. The slides were then washed with PBS and incubated with a HRP-conjugated goat antimouse secondary antibody for 1 h at 37C. An incubation with a freshly prepared 3,3¢-diaminobenzidine (DAB) substrate solution for 5 min at 37C was followed. Slides were counterstained with hematoxylin (Sigma-Aldrich) for 3 min and visualized using a light microscopy (Nikon Eclipse TiSR, Tokyo, Japan). Results and Discussion Sequence analysis of GRAM domain-containing protein 1A

To obtain a suitable antigen for immunization, we analyzed the gene and protein sequence. GRAM domain-containing protein 1A has a complete length of 724 amino acids and contains one GRAM domain (Fig. 1A). The GRAM domain was found in glucosyltransferases, myotubularins, and other putative membrane-associated proteins. It is normally about 70 amino acids in length. It is thought to be an intracellular protein-binding or lipid-binding signaling domain, which might have an important function in membrane-associated processes.(15) We uploaded GRAMD1A reference sequence onto the Protean software program. The data showed that the C-terminus from 191–284 amino acids is highly immunogenic

FIG. 4. (A) Immunoblot analysis of supernatants of subclones with immunogen. Purified recombinant protein GRAMD1A was loaded and electrophoresed on 12% polyacrylamide gel under reducing conditions, transferred to PVDF membrane, probed with MAbs, and visualized with the peroxidase system. Protein concentration loaded in each lane was 50 ng. Lane ( + ), immune sera as positive control; lanes 1–8, supernatants of eight subclones. (B) Western blot analysis of different cell lines using anti-GRAMD1A MAb. The cell lysates were electrophoresed on 8% polyacrylamide gel under reducing conditions, transferred to PVDF membrane, probed with MAb GRAMD1A, and visualized with the peroxidase system. Lane 1, SHG-44; lane 2, NIH/3T3; lane 3, K562; lane 4, HCT116; lane 5, Jurkat.

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GRAMD1A protein was expressed in E. coli strain BL21 under regular condition. We compared the expression level using 0.5 mM and 1 mM IPTG induction. Most GST-tagged GRAMD1A proteins were expressed in a soluble form and there was no significant difference observed between different IPTG concentrations. The recombinant proteins were purified through glutathione sepharose column and the 35 kDa purified protein was confirmed by SDS-PAGE under reduced condition (Fig. 2). Screening of MAbs against GRAMD1A protein

After the fourth immunization, sera from five immunized mouse were tested using ELISA for their reactivity with recombinant GRAMD1A protein. The mouse, which had the highest reactivity with GST-GRAMD1A and a low signal with GST at dilutions of 5 · 10 - 3 to 4 · 10 - 6 (Fig. 3), was selected for fusion. Based on the results obtained from sera, ELISA screening of supernatants was performed by ELISA. ELISA-positive supernatants were further confirmed by immunoblot analysis. The clones that had cross-reactivity with GST were excluded. Stable clones that showed significant reactivity with GST-GRAMD1A but no reactivity with GST were subcloned. After subcloning, clones that reacted strongly with GRAMD1A were acquired and maintained. A total of eight positive clones were confirmed by Western blot analysis (Fig. 4A), and the isotyping of all these clones were IgG1 subtype (data not shown). To evaluate the potential application of eight MAbs in native protein detection, they were tested for Western blot.

SONG ET AL. Novel mouse MAb GRAMD1A can detect native GRAMD1A protein by Western blot analysis

To determine if the newly generated MAbs could detect denatured native GRAMD1A protein, a Western blot analysis was done. Immunoblot of proteins from different cell lysates were separated under reducing conditions. One clone named MAb GRAMD1A specifically recognized a protein that had an apparent molecular mass of 80–90 kDa on NIH/3T3 and SHG-44 cells. The molecular weight of the detected bands corresponds with the data of Uniprot. No bands were present in HCT116, K562, and Jurkat cell lines, which may indicate low levels of endogenous GRAMD1A protein expressed in these cells (Fig. 4B). The screened MAb GRAMD1A was used to examine the expression pattern of GRAMD1A protein for further characterization. GRAMD1A protein expressed in the nucleus of hES and various human carcinoma cell lines

Immunofluorescence assay is used to study the distribution and localization of proteins in tissues and cell lines. To confirm the expression pattern of GRAMD1A protein in human embryonic stem cells, an immunofluorescence assay was performed. The results of immunostaining showed that MAb GRAMD1A specifically localized in the nucleus of hES (Fig. 5B). Undifferentiation marker as SSEA-4 of embryonic stem cell was also present in staining data (Fig. 5A). We then examined the expression of GRAMD1A on various cell lines including human lung adenocarcinoma A549 cells, liver carcinoma HepG2 cells, epithelial carcinoma

FIG. 5. Immunofluorescent staining of hES using MAb SSEA4 (A) and MAb GRAMD1A (B). Lane 1, nuclei staining by Hoechst; lane 2, SSEA-4 and GRAMD1A staining; lane 3, merged images with nuclei staining.

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A431 cells, breast carcinoma SK-BR-3 cells, breast carcinoma MCF-7 cells, colorectal cancer cell line SW480 and HCT116, glioma cells SHG-44, and mouse fibroblasts NIH/ 3T3 using the antibody. The results of immunocytochemistry indicated that the antibody recognized protein GRAMD1A widely expressed in the human cancer cell lines in the nucleus. The staining pattern was similar to the hES staining. In addition, weak staining in the cytoplasm was observed in SKBR-3 and SW480 cells. Positive staining in the nucleus was also found in mouse NIH/3T3 cells. Interestingly, negative staining in human colorectal cancer HCT116 cells may indicate GRAMD1A protein is not strongly expressed in this cell line (Fig. 6). GRAMD1A protein expressed in different human normal and cancer tissues

From the immunofluorescence staining results, we concluded that MAb GRAMD1A specifically stained the nucleus of hES and the nucleus and cytoplasm of different human carcinoma cell lines. We wondered if GRAMD1A protein expressed in human normal and cancer tissues. To investigate its staining pattern, immunohistochemical staining analysis was performed. We analyzed expression of GRAM domain-containing protein 1A in selected tissues of the human reproductive system with MAb GRAMD1A. In the testis, a strong signal of GRAMD1A was seen in the spermiocytes, prespermatid, spermospore, and supporting cells, but not in the testicular interstitial cells. In the breast, intense signal of GRAMD1A was observed in the duct epithelial cells, but not in myoepithelial cells. Significant expression of GRAMD1A was found in the acinous cells of prostate (Fig. 7A). The distribution of GRAMD1A was also analyzed in the human reproductive cancer tissues. GRAMD1A in breast cancer is expressed in a pattern very similar to that of normal breast tissue. In ovarian carcinoma, only diffuse, weak GRAMD1A in the cytoplasm was found in adenocarcinoma cells and staining was not observed in the ovarian interstitial cells. In contrast, intense GRAMD1A signal was observed in the cervical squamous cells of cervical cancer (Fig. 7B). Next we examined the expression of GRAMD1A in human digestive system. In human colon, strong GRAMD1A was observed in the glandular epithelial cell and part of fibroblasts (Fig. 7A). Similar staining was also noted in the adenocarcinoma cells of colon (Fig. 7B). Significant expression of GRAMD1A was found in the acinous cell of pancreas (Fig. 7A). Interestingly, only weak staining was found in the cytoplasm of hepatocyte in liver (Fig. 7A) and GRAMD1A levels were lower in the hepatocellular carcinoma cells (Fig. 7B). Furthermore, moderate staining was observed in gastric adenocarcinoma cells (Fig. 7B). Finally we tested the expression of GRAMD1A in other tissues. In the kidney, medium staining was observed in the renal tubular epithelial cells and partial glomerular endothelial cells. Strong staining was found in the epithelial cells of lung and neurogliocytes of brain (Fig. 7A). A similar pattern was also found in adenocarcinoma cells of lung cancer and clear cells of renal cell cancer. In addition, the intensity of GRAMD1A was largely expressed in the urothelial carcinoma cells (Fig. 7B).

FIG. 6. Immunofluorescent staining of seven human carcinoma cell lines and mouse fibroblasts NIH/3T3 cells using MAb GRAMD1A. Lane 1, nuclei staining by Hoechst; lane 2, GRAMD1A staining; lane 3, merged images with nuclei staining.

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FIG. 7. Immunohistochemical staining of GRAMD1A protein in human normal tissues and human cancer tissues.

MONOCLONAL ANTIBODY AGAINST GRAMD1A Conclusions

Generation of monoclonal antibodies is a key for the investigation of the expression and function of a newly discovered protein in cells and tissues. In our study, one monoclonal antibody specific to human GRAM domaincontaining protein 1A was raised and characterized. MAb GRAMD1A was able to detect GRAMD1A protein in immunocytochemistry and immunohistochemistry, indicating that it is a potentially useful tool in the detection of GRAMD1A in cells and tissues. The establishment of MAb GRAMD1A will be helpful for the future study of dozens of other unknown proteins that were found in the previous study of our group. This method may lead us to discover and identify new biomarkers of hES. Author Disclosure Statement

The authors have no financial interests to disclose. References

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Address correspondence to: Ming Zhang Institute of Genetics Zhejiang University College of Life Sciences 388 Yuhangtang Road Room 309 Hangzhou 310058 China E-mail: [email protected] Received: January 1, 2014 Accepted: April 9, 2014

Production and characterization of a monoclonal antibody against GRAM domain-containing protein 1A.

From the proteomic analysis, we identified hundreds of novel proteins that have never been characterized for their expression profile and function on ...
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