Generation of Mouse FANCL Antibody and Analysis of FANCL Protein Expression Profile in Mouse Tissues

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Acta Genetica Sinica, January

ISSN 0379-4172

2006, 33 (1): 49-55

Generation of Mouse FANCL Antibody and Analysis of FANCL Protein Expression Profile in Mouse Tissues ZHAO Qing-Guo, ZHOU Yu, ZHU Heng-Qi, LU Bai-Songa, HUANG Pei-Tanga Beijing Institute of Biotechnology, Beijing 10007 1, China

Abstract: Fanconi anemia complementation group L (FANCL) is a novel Fanconi anemia protein, which mono-ubiquitinates FANCD2 as a ubiquitin E3 ligase, and plays a crucial role in DNA damage repair and chromosome stability maintenance. FANCL is involved in the proliferation of primordial germ cells (PGC) in early embryonic stages, and may play a role in the development of germ cells by forming a novel testis-specific network with testis-specific proteins in the adult testis. FuncL cDNA sequence was cloned by RT-PCR from mouse testis total RNA, and expressed in E. coli BL21(DE3). Rabbit FANCL polyclonal antiserum was generated using the recombinant protein as the antigen. To prepare an antigen column for affinity purification of FANCL-specific antibody, recombinant His-tagged FANCL was purified by Ni2'-charged HiTrap Chelating HP column and coupled to an NHS-activated HiTrap column. To confirm the activity and specificity of the FANCL antibody, we constructed plasmid pCMV-HAFANCL to transfect HEK 293T cells. Transiently expressed HA-FANCL fusion protein was analyzed by immunoblotting with both the FANCL antibody and HA monoclonal antibody. The antibody was used in Western blotting to check the expression of FANCL protein in mouse tissues. We found wide expression of FANCL in brain, muscle, heart, lung, liver, spleen, kidney, testis, ovary and uterus, indicating the functional importance of this novel protein. Key words: FANCL; antibody; expression profile

Fanconi anemia (FA) is a recessively inherited disease characterized by congenital defects, progressive bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are chromosomally unstable and are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Based on somatic cell fusion studies, FA is comprised of eleven distinct complementation groups. Eight of the FA genes have been cloned and expressed. Studies have demonstrated that FA proteins interact in a common cellular pathway, referred to as the FA pathway, and function at the level of chromatin during S phase to regulate and maintain genomic stability through homologous recombination-mediated DNA repair r1-31. Recently FANCL was found to be a new component of a Fanconi anemia protein core complex, which included 6 other FA proteins FANCA, FANCC, FANCE,

FANCF, FANCG and FANCB'41. It possesses E3 ubiquitin ligase activity and is essential for FANCD2 monoubiquitination '51. In response to DNA damage or DNA replicaton singals, FANCD2 is monoubiquitinated and targeted to nuclear foci containing BRCAl and BRCA2, which participate in homologous recombination. FANCL has a key role in the Fanconi anemia pathway as the catalytic subunit required for monoubiquitination of FANCD2 '6371. The germ cell lineage in the mouse is set aside as primordial germ cells (PGCs) at the early embryonic stage. The germ cell deficient (gcd) mutation is a recessive, transgenic insertional mutation in which a drastic depletion of PGCs occurs in the developing genital ridge, resulting in infertility in both male and female adults. FuncL, once called Pog or PhJ9, was recently reported to be the gene deleted in gcd mice and responsible for the infertile phenotype of the gcd

Received: 2005-03-12; Accepted: 2005-05-15 This Work Was Supported by the National Natural Science Foundation of China(No. 30370718, 304703079).

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Corresponding Author. E-mail: lubaisong @ yahoo.com; peitanghuang @ yahoo,com.cn

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mice. FANCL is critical for normal PGC proliferation in the early embryonic development ", 'I.

Acta Genetica Sinica Vo1.33 No.]

2006

1. 1. 2

FANCL in transient transfected cells. Recent studies

Chemicals and Antibodies Trizol monophasic lysis reagent used for the simultaneous isolation of RNA was purchased from Life Technology Co. Anti-HA antibody (HA-probe HRP) and Western blotting luminol reagent (sc-7392) were obtained from Santa Cmz Biotech Inc. Freund's adjuvant and beta-actin antibody was purchased from Sigma. HiTrap NHS-activated HP affinity column purchased from Amersham Biosciences Co.

found several testis-specific proteins interacting with

1.2

In the adult testis, a novel germ cell-specific gene Ggn (Gametogenetin) was found to encode proteins interact with FANCL. Ggn encoded three putative proteins, GGN1, GGN2 and GGN3 by alternative splicing. Two of them, GGNl and GGN3, interacted specifically with FANCL and co-localized with

GGNl, among these included ornithine decarboxylase-antizyme 3(OAZ3), gametogenetin binding protein 1 (GGNBP1) and GGNBP2. The splicing of mouse Ggnbp2 pre-mRNA in the testis is specific to that in the non-testis tissues. FANCL, GGN1, GGN2, GGN3, GGNBP1, GGNBP2 and OAZ3 proteins probably form a novel testis-specific protein network involved in spermatogenesis

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Given the important role of FANCL in the FA pathway and mouse germ cell development, we prepared rabbit FANCL antiserum and immunoaffimity-purified specific FANCL antibody. It can be used as a specific reagent to further study FANCL in vivo and in

v i m . For example, the specific antibody can be used to determine the precise subcellular location of FANCL, to isolate FANCL protein from tissue homogenates, and to find other macromolecules that interact with FANCL. Here we used the antibody to analyse the expression profile of FANCL in mouse tissues.

1 Materials and Methods 1. 1 1. 1. 1

Materials Cell lines and Animals

HEK 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 2 mmol/L glutamine at 37°C in

Methods

1. 2. 1

Cloning and expression of mouse FuncL cDNA One testis was collected from a male C57BL/6 mouse of 2-month age, and was homogenized in 2 mL of Trizol with a polytron homogenizer. Testis total RNA sample was extracted according to the instruction of the manufacturer. The concentration of the RNA was estimated by measuring the absorbance at 260 nm of an aliquot of the final preparation. First-strand cDNA was synthesized from the testis total RNA with TaKaRa BcaBETTMRNA PCR kit according to the protocol of the kit. In a negative control reaction, the reaction mixture included all components of the first-strand reaction except reverse transcriptase. Coding region of mouse FuncL cDNA was amplified from the mouse testis cDNA using Expand High Fidelity PCR System (Roche) with a pair of specific primers for FancL gene, FancLF: 5'-GGAA'ITCATGGACGAAGCAGAAGCAAG-3' and FancL-R: 5'GGAATTCTCAAGGTTITCTCCCAGAC-3'. The amplified products were analyzed by electrophoresis through a 2.0% agarose gel. The amplified DNA was ligated to the pGEM-T vector to construct pGEM-TFANCL. The identity of the cloned fragments was confirmed by DNA sequencing. pPRoEX-HTaFANCLwas constructed by releasing the EcoR I fragment of FancL cDNA from pGEM-

the presence of 5% COz. Female New Zealand rabbits of about 3 kg body weight and 2-month-old

TRANCL and inserting the fragment to EcoR I site of

C57BL/6 mice were purchased from Beijing laboratory animal center of the Academy of Military Medical Sciences.

pPRoEX-HTa. Theplasmid pPRoEX-Hta /FANCL was transformed into an E. coli BL21(DE3) strain to express His-tagged FANCL.

ZHAO Qing-Guo et al.: Generation ot Mouse FANCL Antibody and Analysis of FANCL Protein Expression Profile in Mouse Tissues

1. 2. 2 Preparation of FANCL rabbit antiserum For large-scale expression and purification of the target protein, 5 mL overnight culture of BL21 (DE3) containing the recombinant were inoculated to 500 mL LB containing ampicillin. The target protein was expressed as an inclusion body with IPTG-inducing for 8 h at 3 0 % . The cells were harvested by centrifugation and disrupted by sonication. The inclusion body was collected by centrifugatin and washed by using Triton X-100

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manufacturer. The FANCL antigen column prepared above was used for immunoaffinity purification of the FANCL antibody from the FANCL rabbit antiserum. The column was washed sequentially with start buffer (10 mmol/L Tris-HC1, pH 7.5) and elution buffer (100 mmol/L glycine, pH 2.5), and was equilibrated with start buffer. The antiserum was diluted 10 X with start buffer and loaded on the column. The bound antibody

pL Freund’s complete adjuvant were used. Three

was eluted with elution buffer (0.1 m o m glycine, pH 2.5). The pH of the eluted antibody was brought neutral by adding 0.1 volume neutralizing buffer (1 m o m Tris-HC1, pH 8.0). 1. 2. 4 Testing of the purified the FANCL antibody activity and specificity with Western blotting pCMV-HA/FANCL was constructed by insertion

successive boosts were followed after the first immu-

of the FancL cDNA into EcoR I site of pCMV-HA.

nization with an interval of 4-6 weeks between each Freund’s incomplete adjuvant were used. Antiserum

The plasmid DNA was transfected into HEK 293T cells with Lipofectamine 2000 to transiently express fusion protein HA-FANCL. Sixty hours after trans-

was collected in 7-10 d after the last boost. Antibody

fection, the cells were collected and resuspended in

titer was measured by enzyme-linked immunoassay (ELIS A). 1. 2. 3 Affinity purification of FANCL antibody The His-tagged FANCL was purified by immobilized Ni2+absorption chromatography, and immobilized to polypropylene for preparation of the FANCL antigen column. The target protein in the inclusion

1 X SDS PAGE gel-loading buffer. Western blotting

and urea. The purified inclusion body containing the target FANCL protein was directly used to immunize female New Zealand rabbit of about 3 kg body weight. For the first immunization, 500 pg antigen and 500

boost. For each boost, 250 pg antigen and 250 pL

body was solubilized with binding buffer (20 mmoVL Phosphate, 0.5 moVL NaC1, 6 moVL guanidium chloride, pH 7.5). The insoluble debris was removed by centrifugation, the supernatant was run through the HiTrap Chelating HP column (Amersham Biosciences) connected to AKTATM purifier (Amersham Biosciences) and was preequilibrated with binding buffer. The protein was eluted with binding buffer containing 500 mmol/L imidazole. The binding buffer of the eluted protein was changed to coupling buffer (0.2 moVL NaHC03, 0.5 mol/L NaC1, pH 8.3, 6 mol/L guanidium chloride) by centrifuging in Centricon 10 (Amicon Co.). The purified protein was immobilized to HiTrap NHS-activated HP affinity column (Amersham Biosciences) according to the

analyses were performed with the purified FANCL antibody and HA monoclonal antibody respectively to test FANCL antibody activity and specificity. 1. 2. 5 Analysis of FANCL protein expression profile in mouse tissues Adult C57BL/6 mice were sacrificed and brain, heart, lung, liver, spleen, kidney, testis, ovary, uterus and muscle tissues were dissected and washed in ice-cold PBS, minced and homogenized at 4°C in ice cold RIPA buffer (150 mmol/L NaC1, 50 mmoVL Tris-HC1 pH 7.5, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate) added with protease inhibitors cocktail from Roche and PMSF from Sigma, and then centrifuged at 10 000 r/min for 30 min at 4°C to obtain clarified lysates. Total protein amounts were quantified using the Bio-Rad protein assay. These lysates were mixed with standard SDS-PAGE gel-loading buffer, heated at 100°C for 5 min, and then separated by gel electrophoresis. The FANCL protein in the mouse tissue was detected by Western blotting with the purified FANCL antibody.

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2 Results and Discussion 2. 1

Mouse FancL cDNA was cloned and expressed in E. coli

2006

His-tagged FANCL protein(- 44 kDa) was visualized by staining with Coomassie Brilliant Blue (Fig. 2). 1 2 3 k D a -91

To prepare the FANCL protein as antigen, mouse FuncL cDNA was cloned and expressed in

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E. coli. We extracted mouse testis total RNA sample

with Trizol, and synthesized first-strand cDNA in a reaction catalyzed by M-MLV reverse transcriptase and primed by oligo(dT)15.Coding region of mouse FancL cDNA was amplified from the first-strand cDNA with a pair of specific primers for FancL gene. The amplified product was analyzed on an agarose gel stained with ethidium bromide to visualize the DNA. and a 1.1 kb DNA fragment of the expected size was yielded (Fig.1). We constructed pGEM-TFANCL for sequencing and subsequent manipulation by inserting the DNA fragment into pGEM-T vector. We then confirmed the identity of the cloned FuncL cDNA fragment by DNA sequencing. pPRoEX-HTa/FANCL was constructed by releasing the FancL cDNA fragment from

Fig. 2 FANCL expressed in E. coli. I: IPTG-induce expression of FANCL(arrowhead) for 2 h; 2: An uninduced sample was used as a negative control; 3: Protein molecular weight marker.

2.2

A FANCL rabbit antiserum was obtained

To prepare FANCL protein used as antigen for injection, we expressed His-tagged FANCL protein on a large scale in E. coli induced with 1 mmol/L IPTG at 30°C for 8 h. The bacterial cells were disrupted by sonication and the inclusion body was col-

pGEM-T/FANCL and inserting into EcoR I sites of

lected by centrifugation at 12 000 r/min, 4°C for 15

pPRoEX-HTa.

min, washed with Triton X-100 and urea to remove as much bacterial protein as possible. The proteins in the inclusion body were solubilized directly in 1xSDS gel-loading buffer and analyzed by SDS-PAGE to characterize the His-tagged FANCL protein purity. We have used a Shimadzu (Tokyo, Japan) CS-9301 PC densitometer for densitometric scanning of the washed inclusion body. SDS-PAGE showed the His-tagged FANCL protein purity close to 85%.

Fig. 1 Amplification of FuncL cDNA generated by reverse transcription of mouse testis total RNA 1: DNA size marker; 2: FuncL cDNA fragment (1.1 kb) amplified; 3: A negative control.

The His-tagged FANCL protein in the washed inclusion body was directly used as antigen to immu nize female New Zealand rabbit.Seven to ten days after the third boost, a test bleed was taken and used to obtain a measure of antibody titer, which was cal-

pPRoEX-HTaANCL was transformed into the E. coli BL21(DE3) strain. Log phase culture was in-

culated to be 1:16 000. We began to bleed the rabbit every week thereafter. Each bleed was assayed indi-

duced with 1 mmol/L IPTG at 30°C. Cells were col-

vidually, when the antibody titer started to fall, the

lected by centrifugation, lysed directly in 1X SDS gel-loading buffer, and boiled for 3 min. Proteins in the

rabbit was terminally exsanguinated by cardiac puncture. We obtained about 50 mL of the antiserum

cells were separated by SDS-PAGE, and the induced

altogether.

ZHAO Oine-Guo et al.: Generation of Mouse FANCL Antibody and Analysis of FANCL Protein Expression Profile in Mouse Tissues

The specific FANCL antibody was purified by affinity chromatography

2. 3

To obtain high purity FANCL protein for preparing a FANCL antigen column, we purified the His-tagged FANCL protein from the washed inclusion body by immobilized Ni2+ absorption chromatography with AKTATMpurifier(Fig.3). The purified protein (1-5 mg) was immobilized to 1 mL HiTrap NHS-activated HP column, the FANCL antigen column was prepared as described above, and used for affinity purification of the FANCL antibody from the rabbit antiserum. One mL (0.5 mg/mL) FANCL antibody was purified from 5 mL the rabbit antiserum on the antigen column with excellent results (Fig. 4). We

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The activity and specificity of the purified antibody was further tested by Western blotting analysis. We transiently expressed fusion protein HA-FANCL by transfecting pCMV-HA/FANCL plasmid into HEK 293T cells, We loaded the lysate of cells containing recombinant HA-FANCL as target and the lysate of cells transfected by pCMV-HA plasmid as a negative control. Western blots were performed using the purified FANCL antibody and HA monoclonal antibody at a dilution of 1500 and 1:2500 respectively. Fig. 5 shows the Western blot results from the two antibodies. HA-FANCL is visible as a band at about 43 kDa size in both blots probe with FANCL antibody and HA antibody. The purified FANCL antibody recongnized recombinant FANCL with excellent activity and specificity. WB: FANCL

checked and collected the tubes containing the

kDa

-

+

WB: HA -

+

HA-FANCL

FANCL antibody by Western blotting. The antibody was of high purity as analyzed by SDS-PAGE. kDa

1

2

3

t

4

36-

Fig. 3 SDS-PAGE of purified His-tagged FANCL by immobilized Ni2+absorption chromatography 1: Molecular weight marker; 2: 1 mg/mL BSA (asterisk); 3: 2 mg/mL BSA; 4: Purified FANCL (arrow). 2 and 3 were used as an quantitative standard.

A200 5.0

Washinn buffer

-I

4.0

Elution

t eer ~

FANCL antibody

-

Fig. 5 An assay of FANCL antibody activity and specificity by Western blotting HA-FANCL transiently expressed from HEK 293T cells is analyzed by immunoblotting with FANCL antibody and HA antibody to demonstrate the activity and specificity of the antibody. Both FANCL and HA antibodies detected a band of about 43 kDa (arrow). A band label by an asterisk was detected by FANCL antibody, Which could be result of recombinant HA-FANCL degradation.

2.4

Widespread expression of FANCL protein in mouse tissues To check the expression profile of FANCL at

3.0

the protein level in mouse tissues, we prepared and affinity purified specific FANCL antibody as de-

2.0

scribed above. We prepared various tissue lysates including brain, heart, lung, liver, spleen, kidney,

1.o \

150

200

250 mL

Fig. 4 Purification of FANCL antibody from rabbit antiserum on the FANCL antigen column

testis, ovary, uterus and muscle, and applied the antibody to these tissue lysates in Western blotting to check the expression of FANCL. We could detect FANCL protein with an apparent molecular

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Vo1.33 No.1 2006

weight of 41 kDa as expected in all these tissue

Cell Cycle, 2005,4(1) : 80-86.

lysates (Fig. 6). The result shows FANCL protein is

Timmers C, Taniguchi T, Hejna J, Reifsteck C, Lucas L, Bruun D, Thayer M, Cox B, Olson S, D'Andrea A D, Moses R, Grompe M. Positional cloning of a novel Fanconi anemia gene, FANCD2. Molecular Cell,

widely expressed in various mouse tissues.

2001, 7 : 241-248. FANCL

"rw)

@p-actin

Meetei A R, Winter J P, Medhurst A L, Wallisch M, Waisfisz Q,van de Vrugt H J, Oostra A B, Yan Z, Ling C, Bishop C E, Hoatlin M E, Joenje H, Wang W. A novel ubiquitin ligase is deficient in Fanconi anemia. Nature Genetics, 2003, 35(2) : 165-170.

Fig. 6 Analysis of mouse tissue expression profile by Western blotting FANCL protein was detected in uterus, ovary, testis, kidney, spleen, liver and lung tissue (from left to right), in addition to

Meetei A R, Yan Z, Wang W. FANCL replaces BRCA 1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination. Cell Cycle, 2004, 3(2) : 179-181.

We have described the preparation and affin-

Agoulnik A I, LU B, Zhu Q, Truong C, Ty M T, Arango N, Chada K K, Bioshop C E. A novel gene, Pog, is necessary for primordial germ cell proliferation in the mouse and underlies the germ cell deficient mutant, gcd. Human Molecular Genetics, 2002, 11 : 3047-3053.

ity purification of a rabbit polyclonal FANCL an-

Pellas T C, Ramachandran B, Duncan M, Pan S S,

tibody and demonstrated that it is of high speci-

Marone M, Chada K. Germ-cell deficient(gcd), an inser-

brain, muscle and heart tissue (data not shown). p-actin protein detected by Western blotting was used as an internal standard.

ficity and is an excellent tool for Western blot analysis. We have applied the antibody to various mouse tissue lysates in Western blotting to check the expression of FANCL protein. We have found that FANCL is wide expressed in mouse tissues. This highlights its biological importance. The an-

tional mutation manifested an infertility in transgenic mice. Proceedings of the National Academy of Sciences of the United States ofAmerica, 1991, 88 : 8787-8791.

[lo] Zhang J, Wang Y, Zhou Y, Cao Z, Huang P, Lu B. Yeast two-hybrid screens imply that GGNBP1, GGNBP2 and OAZ3 are potential interactionpartners of testicular germ cell-spedic protein GGN1. FEBSLetters,2005,579 : 559-566.

tibody should provide an important tool for future studies to elucidate the precise biological roles of FANCL.

Zhou Y, Zhao Q, Bishop C E, Huang P, Lu B. Identification and characterization of a novel germ cell-specific gene Ggnbp 1. Molecular Reproduction Development,

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2005,70 : 301-307. gain of fertility in POG-deficient mice indicate that POG is not necessary for the proliferation of spermatogonia. Biology of Reproduction, 2003,69 : 161-168. LU B, Bishop C E. Mouse GGNl and GGN3, two germ cell-specific proteins from the single gene Ggn, interact with mouse POG and play a role in spermatogenesis. The Journal of Biological Chemistry, 2003,278(18) : 16289-16296. [14] Zhao Q, Zhou Y, Cao Z, Zhu H, Huang P, Lu B. Germ-cell specific gametogenetin protein 2 (GGN2), expression in the testis, and association with intracellular membrane. Molecular Reproduction Development, 2005, 72(1) : 31-39.

ZHAO Qing-Guo et al.: Generation of Mouse FANCL Antibody and Analysis of FANCL Protein Expression Profile in Mouse Tissues

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