Newsletter

Biochemical analysis of HLA-DR antigens using one-dimensional isoelectric focusing gel electrophoresis u

R. Ishizuka, T. Kaidoh, T. Matsuo, Y. Koide, T. 0. Yoshida. Biochemical analysis of HLA-DR antigens using one-dimensional isoelectric focusing gel electrophoresis. Tissue Antigens 1992: 39: 220-224.

u

R. Ishizuka, T. Kaidoh, T. Matsua, Y.Koide and T. 0. Yoshida Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Hamamatsu. Japan

Key words: D typing - HLA class II antigens one-dimensional isoelectric focusing gel electrophoresis - PCR/dot blot hybridization - iwodimensional gel electrophoresis Received 31 October, accepted for publication 27 January 1992

At the 10th International Histocompatibility Workshop in 1987, biochemical analyses of HLA class I1 and class I antigens were performed by the two-dimensional gel electrophoresis system (2DGES) (1, 2) and the one-dimensional isoelectric focusing (1 D-IEF) gel electrophoresis (3-5), respectively, leading to findings of a number of new variants (splits, subtypes). It was scheduled for us to host the biochemistry component of the 1lth International Histocompatibility Workshop in 1991. For the use in the component, we have attempted to improve the procedure of ID-IEF to analyze HLA class TI antigens, making it simpler and easier to compare each band. Cells employed were 6 1 Epstein-Barr virus-transformed B-cell lines of the 1 1th International Histocompatibility Workshop panel cells, 6 DR4 homozygous B-cell lines and 15 DR4 or DR9 heterozygous Japanese B-cell lines. High frequency of DR4 and DR9 antigens in Japanese led us to employ DR4 and DR9 B-cell lines. A monoclonal antibody NC-I was used to precipitate DR antigens, while PLM-12 (6, 7) and PLM-13 (7) were used to precipitate DQ antigens. As shown in Fig. 1 (5), 1-2 x lo' cells of human B-cell lines were prepared for the DR antigen analysis and 3 4 x lo7 cells for the DQ antigen analysis. These cells were precultured in methionine-free RPMI 1640 medium (Sigma) for 2 h, and 220

then further cultured with 0.1 mCi of "Smethionhe/ 1 x lo7 cells for 14 h. The cells were then lysed on ice using 1 ml of 0.5% Triton X-100 lysis buffer (lysis buffer:lO mM Tris. HCI (pH 7.4), 140 mM NaCI, 0.5% Triton X-100). The lysate was centrifuged at 9000 rpm for 20 min and the supernatant was applied to immunoprecipitation. Immunoprecipitation was performed by protein A-sepharose (20 pg/20 pl of packed swollen sepharose) conjugated with polyclonal rabbit-antimouse IgG (H+L) bound with an anti-class I1 monoclonal antibody. Before immunoprecipitation, the lysate was pre-cleared by protein A-sepharose conjugated with polyclonal rabbit-anti-mouse IgG (1 ml of lysatel20 pl of the protein A-sepharose). The class I1 antigen-bound sepharose was washed repeatedly. Sialic acid was removed with neuraminidase type V (Sigma) at 37°C for 90 min. Then class I1 antigen was released by sample buffer (sample buffer: 8.0 M urea, 2% ampholine, 2% Nonidet P-40, 5% 2-mercaptoethanol) at 50°C for 45 min, centrifuged at 15000 rpm for 1 min, after which the supernatant was applied to the 1D-IEF. IEF gel contained 4.5% acrylamide (29.2% w/v acrylamide and 0.8% w/v bisacrylamide), 1.7% Nonidet P-40, 8 M urea, 3% ampholine (LKB) (mixture for DRP-chain gels: pH 3.5-10, pH6-8; 2:l v/v: mixture for DQP-chain gels: pH 3.5-10, pH 4-6, pH 5-8; 2:1.5:1.5 v/v/v), 0.04% am-

Newsletter

1D-IEF Cells : EBV-transformed B-cell line (over 1 x 10')

1.

+

Metnbolic labeling using 35S-rnethionine Lysis or labeled cells

I

Protein A-sepharose with rabbit anti m o u s e IgG

1 7 t

InimunoprecipitPtion

-

1 i

Neurnniinidase treatment

ID-IEF

I

t

Flnurography

Figure I. Steps of the protocol of one-dimensional isoelectric focusing ( I D-1EF)gel electrophoresis improved for HLA-DR antigen analysis.

monium per sulfate, and TEMED. The sample (2 x lo4 cpm/well) was applied from the anode. We performed the 1D-IEF using a vertical-type apparatus with coolant circulation tank (Resolmax, ATTO) to avoid the temperature increase during electrophoresis. Electrophoresis was carried out for 18 h at an initial current of 5 mA and a maximum voltage of 800 Y The proteins were fixed for 60 min with 200 ml of methanolfacetic acidlwater (40:lO:lOO vlvf v) after completion of electrophoresis. The gel was dehydrated for 20 min in DMSO and then immersed in 20% PPO in DMSO for 10 min. After washing with tap water for 1 h, the gel was dried and exposed to an X-ray film at -75°C for 3 d. A marker (8, 9) was obtained by labeling horse myoglobin with 14C before carbamylation. More than 13 bands from pH 7.2 to 5.2 were derived from this marker. We analyzed 22 homozygous B-cell lines which were typed DR4 serologically. DRP-chains of DR4 appeared to be classified into 4 bands with different PI values, namely, 4.1, 4.2, 4.3, and 4.4 (Fig. 2). DRP-chains of DRw8 were classified into bands with 8.1 and 8.2, DRwll with 11.1 and 11.2 (Fig. 3), DRwl4 (DRw6-associated) with 14.1 and 14.2 and DRwl5 (DR2-associated) with 2.12 and 2.2. DRP-chain of DRI, DR7, DR9, DRwl2 (DR5associated), DRw 13 (DRw6-associated), and

lD-IEF(DR4)

I D-lEFtDR)

M

1 2 3 4 5 6 7 8 9 1 0

M

pH

-8 Mom Ab : NC-1

Mono Ab : NC-1 1)FS 2)L-KT13 3)Wa 4)LeolO5)~AS156k-KT17 7)TSlO (DR4.3XDR4.2XDFI4.4XD.3) (DR4.4) (DR4.2) (DR4.31 D W W DWlO D#KK?Ik*15hWlO Dw15 DwKT2 DwlO

8)YAR SSUD 10)JAH M)Ma*W (DR4.3XDR4.11 (DR4.11 Dlyphp DWlO Dw4 cw4

Figure 2. HLA-DR antigens of homozygous B-cell lines serologically typed as HLA-DR4 were differentiated by verticaltype ID-IEF into 4 subtypes of DR4.1, DR4.2, DR4.3 and DR4.4. D types are shown in parentheses. The monoclonal antibody used was NC-I. Ampholine used was in the range pH 3.5-10.

1IOLGA(0LL) 2)MADURA 3)SAVC 4)TISI 5IBM21

cetla

(DR8.2) (DR8.1) (DR4.1XDR11.2)~DRll.l~ hnr8.2 m.1 Dw4 DNew Dw5 6IBM16 7)Sa MIMarker (DRwl2)(DRl) DB6 D?

ID-IEF DWW cells ID-IEF DIWX

Figure 3. HLA-DR antigens of homozygous B-cell lines serologically typed DRw8, DRwll and DRwl2 were analyzed by vertical-type ID-IEF. DR1 and DR4 antigens were electrophoresed on the same gel. The monoclonal antibody used was NC1. Ampholine used was in the range pH 3.5-10.

221

Ishizuka et al. lPIEF(M) beta)

MamA3:W-l

on -5

Table 2. Correlation of the 1C-IEF-identified DR antigens except OR4 subtypes with D types ~

Cell name

* 4

DR1-associated

.5

Owl

sa

Dwl Owl Dwl Dw20 ~ ~

t

-

I

-

LWAGS

-7

-

I -

~z0707a DR2-associated

-8

(-)

shown on the right margin. The marker of horse myoglobinlabeled I4Cis shown on the left margin. f represents anode. represents cathode. The monoclonal antibody used was NC-1. DR3-associated

DRw 16 (DRZ-associated) showed a single band with each PI value (data not shown). DRP-chain of DR3, DRw 17 (DR3-associated), and DRw 18 (DR3-associated) showed an indistinguishable band with same PI value (data not shown). The correlations of these bands are illustrated in Fig 4. These results were in agreement with classification by the 2D-GES at the 10th International Histocompatibility Workshop (10, 11). However, compared to ID-IEF, 2D-GES was laborious and technically complicated. The procedure of our vertical-type

DRS-associated

DRw6-associated

Table 1. Correlation of the DR4 subtypes identified by 1D-IEF with D types Cell name

Dw

DR4.1

BM14 SAVC DEU SUD JAH PF97387 EM92 MT14B THO

Dw4 Dw4 Dw4 Dw4 Ow4 Dw4 Dwl4 Dwl4 Dwl4

OR4.2

L-KTl3 L-KT17 JHAF

DwKT2 DwKT2 Dwl3

DR4.3

YAR Leo10 TS10 FS 2046

DwlO

HAS15 L-KT3 L-KT9

Dw15 Owl 5 Dwl5 Owl 5

DR4.4

v\ra

Dwl 0 Owl 0 Dwl 0 Dwl 0

DRwBassociated

DRSassociated

~~

DR1 DR1 OR1 DR1 DRl 0 DR1

2

D?

STElNLlN DUCAF RSH

D? Dw3 DRSH

DR3 DR3 DR3

EM21 SPOOlO JVM TlSl BM16

Dw5 082 DNew DNew DB6

OR1 1.1 DRl 1.I OR11.2 DRl DR!

CBG6B HHK wr47 TEM LZL AMALA

Dwl8 Dwl8 Owl 9 Dw9 Dwl6 Dwl6

OR#

BER OBB MOU PITOUT BH PLH

Dw7 Owl 1 Dw17 Dwl7 DE1 DB1

DR7

MADURA LUY TAB089 OLGA

0~8.1 Dw8.3 Dw8.3 Dw8.2

DtIY. DR8.1 DR8.2

OK8

Dw23 Dw23 Ow23 D? D?

DR9 DR9 OR9 DR9 DRS/DRwl2

HID L-KTl2 ALBO ' T7527

Dw2 Ow2 Dw2 Dwi2 Ow21 Dw22

~

1D-IEF DR beta

DR2.2 DR2.2 DR2.2 DR2.12 DR2.16 DR2.16 DR2.2

~

DR7-associated

1-D DR beta

wra MGAR AMAl ~4181324 KASOl1 RML H0104

Figure 4. The correlation among DRP-specific bands. pH is

222

WTlOObis JESTOM HOM2

4 -

~

Dw

.

\ti 3

DI

I, D!

r

ID-IEF system was shown to be technically simple and highly reproducible and it was easier to compare the bands of multiple samples on the same gel. Correlation of the DR4 subtypes identified by the ID-IEF with the D types was investigated. The band of DR4.1 was identified from the band of cells which was typed Dw4 and Dw14, DR4.2 was DwKT2 and Dw13, DR4.3 was DwlO and DR4.4

Newsletter Table 3. ORB1 alleles of the DR4 homozygous B-cell lines. The protocol of DNA analysis was performed by the method of the 1l t h International Histocornpatibility Wrkshop Dw

DRBl alleles

Serology DRw52l53

DR4.1 OR4.1 DR4.1 DR4.1 DR4.1 OR4.1 DR4.1 OR4.1

Dw4 Dw4 ow4 Dw4 Ow4 Dwl4 Dwl4 Owl 4

0401 0401 0401 0401 0401 0404 0404 0404 ,

Dw53 Dw53 Ow53 Dw53 Ow53 Dw53 Ow53

JHAF

DR4.2

Owl 3

0407

Ow53

YAR

DR4.3 DR4.3 DR4.3

Dwl 0 Dwl 0 DwlO

0402 0402 0402

Owl 5 Dwl5 Owl 5

0405 0405 0405

Cell

1-0

name

OR beta

BM14 SAM: DEU JAH PF97387 BM92 M i l 4B THO

FS

046 .AS15 ,KT3

DR4.4 DR4.4 DR4.4

\Na

-

Dw53 Ow53

Table 4. Analysis of Japanese DR4 and OR9 B-cell lines by 1DIEF. DR antigens were more definitely classified into each subtype

Cell name

Serology DR

1-D DR beta

Yoneyama Takeshita H. Takeshita F. Takimoto EEDYA EEKAG EEKUN EENUR EETKE EEDEH EElX EEYOS EEHAG KOBA EB-NlS

DR4.1IDR8.2 DR4.2lDR9 DR4.ZDR2 DR4.2/? DR4/? DR4/? DR4/DR2 DR4/0RwG DR4/DR9 DR4.llORl DR4.1/DRw8 DR41DR2 DR9/? DR9/DRw8 DR9/DRw15

DR4.1/DR8.2 DR4.ZOR9 OR4.2/DR2.12 OR4.2lDR4.2 DR4.2lDR4.2 DR4.4lDR8.1 OR4.2lDR2.12 OR4.1/ORw13 DR4.110R9 OR4.1/OR1 OR4.1 IDR8.1 OR4.2/0R2.2 DR9/DR9 OR9/OR8.1 DR9/DR2.2

Furthermore, the DR4 homozygous B-cell lines were analyzed by PCR/dot blot hybridization. DRBl of DR4.1, DR4.2, DR4.3, and DR4.4 ap-

5 (Table 1). Each band of the other DR

Showed complete concordance with D iwn in Table 2.

1D-IEF(DR)

,-. 1 =%DR)

f

M

1 2 3 4 5 6 7 8 S10111213M

1 2 3 4 5 8 7 8

I.

-i

L:

'--5

-8 Mono Ab : NC- 1

Mono Ab : NC-1

bib 1MV

6)EBV-OYA 7)TMMOTO 8)EBV-TKE Q)LeolO lO)L-KT17 (DR4.2) (DR4.2) (DR4.11DRg) (DR4.3) (DR4.2)

MIS iMEF Dtrpino

1)ARBO 2)DKB 3)T7527 4)HID (DRQ/DRQ) (DRQ/DRQ) (DRQ/DRwl2) (DW/DW) D? Dw23 D? Dw23

5)RLO(E6061) 6)C.Wong 7)L-KT12 812046 (DR4.3/DR4.1) (DRQ/DRQ) (DFWDRQ) (DR4.3lDR4.3) DtypHIO hnlO/D? DB5 Dw23 DWlO M)Marker : Extra band IDEF

Figure 5. Japanese heterozygous 9-cell lines were analyzed by vertical-type 1D-IEE DR4, DRw8 and DR9 homozygous Bcell lines were electrophoresed on the same gel. The heterozygous 9-cell lines showed two bands. The monoclonal antibody used was NC-I. Ampholine used was in the range pH 3.5-10 combined with range pH 6-8.

Figure 6. Two cell lines, T7527 and L-KT12, show extra bands. T7527 was typed DR9JDRwl2 and L-KT12 was typed DR9/ DR9 by serological typing and DNA typing. + represents extra band. The extra band of L-KT12 showed almost the same position as DR4.3. The monoclonal antibody used was NC-I. Ampholine used was in the range pH 3.5-10.

223

Ishizuka et a]. peared to be 0401 and 0404,0407,0402, and 0405, respectively (Table 3) (1 1). The 12 Japanese DR4 heterozygous B-cell lines and 5 DR9 heterozygous B-cell lines were analyzed by the vertical-type 1D-IEF (Fig. 5). The results indicated that the DR antigens were more definitely classified into each subtype (Table 4). Among all results of 1D-IEF, two cell lines T7527 and L-KT12 - showed unexpected extra bands (Fig. 6). T7527 was typed DR9/DRwl2 and L-KT12 was typed DR9/DR9 by serological typing and DNA typing. T7527, however, showed a definite extra band in addition to Dq9 and DRwl2 bands, and L-KT12 showed an extra band at almost same band as DR4.3 in addition to DR9 band. To characterize the extra bands, we performed 2D-GES. The results revealed that these extra bands were derived neither from DRa-chain nor from DRP-chain (data not shown). The improved procedure of the vertical-type 1DIEF technique employed here permits of all DRPchains. For DQa- and DQP-chains (12), however, the results of biochemical analysis obtained by 1DIEF were as follows (data not shown): (1) Although the specific bands for DQP-chain were observed by the 1D-IEF, some other nonspecific bands were also observed. (2) No band of DQa-chain was identified. (3) Analyses of DQ antigens were attempted by "'I for labeling of cell surface antigens, but discrimination of bands failed. Since IEF detects only charge-differences, it does not always distinguish every polymorphism of molecules. Nevertheless, IEF can define variants of HLA antigens, that are not yet recognized by serology, and it provides a better estimate of the extent of polymorphism. Acknowledgments

The authors would like to thank Dr. Hiroo Maeda for gifts of monoclonal antibodies NC-1, PLM-12 and PLM-13, Dr. Sonoda and Dr. Yashiki for DR4 and DR9 Japanese heterozygous B-cell lines.

224

References 1. Knowles RW, Kiebsle B. Standardised equipment, reagents, and techniques developed for the 10th workshop 2-D gel study of HLA class I1 antigen. In: Dupont B, ed. Immunobiology of HLA, vol. 1. Histocompatibility Testing 1987: 425-7. 2. O'Farrell PH. High resolution two-dimensional electrophoresis of protein. J Biol Chem 1975: 250: 4007-21. 3. Yang SY. Nomenclature for HLA-A and HLA-B alleles detected by one-dimensional isoelectric focusing (I D-l EF) gel electrophoresis. In: Dupont B, ed. Immunobiology of HLA, vol. 1 Histocompatibility Testing 1987: 54-7. 4. Yang SY. A standardized for method for detection of HLAA and HLA-B alleles by one-dimensional isoelectric focusing (IEF) gel electrophoresis. In: Dupont B, ed. Immunobiology of HLA, vol. 1. Histocompatibility Testing 1987: 332-5. 5. Rodriguez de Cordova S, Numez-Roldan A. Molecular characterization by high-resolution isoelectric focusing of the products encoded by the class I1 region loci of the major histocompatibility complex in humans. I. DR and DQ gene variants. Hum Immunol 1987: 20: 71-93. 6. Marsh SGE, Bodmer JG. HLA-DR and DQ epitopes and monoclonal antibody specificity. Immunol Today 1989: 1 0 305-13. 7. Maeda H, Hirata R. Multiple epitope on a single DQ molecule from the DQw3-carrying haplotypes. Tissue Anfigens 1986: 28: 136-45. 8. Dottavio-Martin D, Ravel JM. Radiolabeling of proteins by reductive alkylation with ["C] formaldehyde and sodium cyanoborohydride. Anulyt Biochem 1978: 87: 562-3. 9. Anderson NL, Hickman BJ. XXIV isoelectric point standards for two-dimensional electrophoresis. Anal-vt Biochem 1979: 93: 312-20. 10. Knowles RW. Nomenclature for two-dimensional (2-D) gel patterns of the HLA class I1 CI and p chains defined in the tenth workshop. In: Dupont B, ed. Immunobiology of HLA, vol. I. Histocompatibility Testing 1987: 58-61. 11. Bodmer JG, Marsh SGE. Nomenclature for Factors of the HLA system, 1990. Tissue Antigens 1991: 37: 97-104. 12. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 1981: 256: 1604-7.

Address: R. Ishizuka Department of Microbiology and Immunology Harnamatsu University School of Medicine 3600 Handa-cho Hamamatsu 431-31 Japan