4 HLA Laboratory, American Red Cross Services, 812 Huntington Avenue, Boston, MA 02115, USA. Polymorphic residues in the N terminal domains of major.
h lBllnO-
lmmunogenetics 29: 346-349, 1989
genetics
© Springer-Verlag 1989
Brief communications Sequence polymorphism of HLA-DP beta chains
Janet S. Lee 1, Silvia Sartoris 2., Paola Briata it, Ed Choi 2, Constance Cullen 2, Denis Lepaslier 3, and Ivan Yunis 4
1 Immunology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA 2 Department of Microbiology and Molecular Genetics, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, OH 45267-0524, USA 3 Centre d'etude Polymorphisme Humaine, 3, rue d'Ulm, F-75005 Paris, France 4 HLA Laboratory, American Red Cross Services, 812 Huntington Avenue, Boston, MA 02115, USA
Polymorphic residues in the N terminal domains of major histocompatibility complex class I113 and to a lesser extent class II c~ chains influence interactions of the molecules with peptide antigens and T-cell receptors, and are the basis for HLA restriction of effector to target cell interactions (Korman et al. 1985). While first domains of several HLA-DRB1, DRB4, DQA1, and DQB1 alleles have been sequenced and analyzed, much less is known about sequence polymorphism of the HLA-DPA1 (hereafter referred to as DPA1, nomenclature according to the Nomenclature Committee on Leukocyte Antigens, 1988) and DPB1 genes. In addition, polymorphism detected at the DP locus by cellular assays is much lower than that for DR and DQ (Wank and Schendel 1984). We undertook DNA sequences analysis of HLA class II cDNAs from HLA homozygous cell lines to define the polymorphisms within allelic groups more precisely. Full-length cDNA clones encoding DPB1 chains were obtained from four cell lines that were analyzed for the Tenth International HLA Workshop. These clones are available on request. HLA homozygous Epstein-Barr virus (EBV)-transformed B-cell lines were obtained from Drs. S. Y. Yang and B. Dupont, for the Tenth International Histocompatibility Workshop, New York, 1987 and maintained in standard conditions. RNA was extracted from the cell lines VAVY, RSH (not consanguineous), BH, and DBB (Workshops 9021, 9023, 9046, and 9052), polyadenylated RNA was selected, and cDNA libraries were generatred as described (Sartoris et al. 1987). cDNA clones were isolated using specific oligonucleotide probes. Inserts from several cDNA clones were subcloned into M 13 and sequencing was performed by the chain termina-
Present addresses: *lstituto di Scienze lmmunologiche, Policlinico di Borgo Roma, 1-37100 Verona, Italy and *Clinica Medica RR, Istituto Scientifico di Medicina Interna, Viale Benedetto XV 6, 1-16121 Genova, Italy Address correspondence and offprint requests' to: J. S, Lee
tion method (Sanger et al. 1977) with Sequenase from the US Biochemical Co. (Cleveland, Ohio). Universal primers and class II B-specific primers (Bell et al. 1987) were used. The DNA sequences derived in this study for two DPwl DPB1 cDNA clones and one DPw4 DPB1 cDNA clone are shown in Figure 1. It is apparent from the sequences that the DPwlb DPB1 cDNA clone isolated from the cell line RSH is very different from the DPwla DPB1 cDNA clone from the cell line VAVY, even though both cell lines were typed as DPwl by three independent laboratories during the Tenth International HLA Workshop (Mickelson et al. 1989). Comparison of the sequences with those for two other cDNA clones that we analyzed indicates that DPwlb is more closely related to DPw4 than to DPwla. This is true not only for the second exon corresponding to the first domain of the protein, but also for the rest of the coding region and the 3' untranslated region, even though some minor differences between sequences in these regions can be found. Analysis of restriction fragment polymorphism of RSH within the Tenth International HLA Workshop indicated that this cell line was heterozygous at the DPA1 and DPB1 loci (Simons et al. 1989). In order to ascertain whether RSH actually expresses a DPwla DPB1 allele, RNA from the RSH cell line was subjected to amplification by the polymerase chain reaction (PCR) procedure (Mullis and Faloona 1987) using specific primers from exon i and exon 3. The PCR products were gel purified and sequenced using labeled primers and TaqTrack (Promega, Madison, Wisconsin). The data are consistent with the presence, at least for the N terminal domain, of the DPwla DPB1 allele as found in the cell line VAVY, and the DPwlb DPB1 allele represented by the cDNA clone that we sequenced (data not shown). Thus, this sequence represents a "blank" allele even though it is closely related to other alleles. When amino acid sequences for several known DPB1 alleles are compared, the differences between the
J.S. Lee et aI. : Sequence polymorphism of HLA-DP beta chains
347
DPwla DPwlb DPw4
TTTTTTTTTG
CCATCCTTTT
exon CCAGCTCCAT
1 GATGGTTCTG
CAGGTTTCTG
CGGCCCCCCG
60
DPwla DPwlb DPw4
GACAGTGGCT
CTGACGGCGT
ex0n TACTGATGGT
1 GCTGCTCACA
TCTGTGGTCC
AGGGCAGGGC
120
DPwla DPwlb DPw4
........................................................... v CACTCCAGAG
AATTACGTGT ---C-TC-TC ................ C-T-
exon 2 ACCAGGGACG GCAGGAATGC TACGCGTTTA ATGGGACACA T ....................................... T .......................................
180
DPwla DPwlb DPw4
exon 2 GCGCTTCCTG GAGAGATACA TCTACAACCG GGAGGAGTAC GCGCGCTTCG ACAGCGACGT ...................................... T- -T .................. ...................................... T .....................
DPwla DPwlb DPw4
exon 2 GGGGGAGTTC CGGGCGGTGA CGGAGCTGGG GCGGCCTGCT GCGGAGTACT GGAACAGCCA ...................................... A- -A .................. ............................................................
nPwla DPwlb DPw4
exon 2 GAAGGACATC CTGGAGGAGA AGCGGGCAGT GCCGGACAGG ........................................ ........................................
GTATGCAGAC ACAACTACGA A-G ................. A-G .................
360
DPwla DPwlb DPw4
GCTGGACGAG GCCGTGACCC TGCAGCGCCG A~TCCAGCCT ..... G--G-C--A ........................... ..... G--GC--A ...........................
AAGGTGAACG TTTCCCCCTC G ...... T ........... G ...... T ...........
420
DPwla DPwlb DPw4
exon 3 CAAGAAGGGG CCCCTGCAGC ACCACAACCT GCTTGTCTGC CACGTGACAG ATTTCTACCC . . . . . . . . . . . . . . . . .T. . . - ........................... G .......... ............. T .................................. G ...........
DPwla DPwlb DPw4
exon 3 AGGCAGCATT CAAGTCCGAT GGTTCCTGAA TGGACAGGAG GAAACAGCTG ............................................................ .............................................................
DPwla DPwlb DPW4
exon CACCAACCTG ATCCGTAATG GAGACTGGAC ............................... ...............................
DPwla DPwlb DPw4
GGGTCGTGTC
3 CATCCAGATC CTGGTGATGC TGGAAATGAC T ............................ T ............................
exon 3 GGAGACGTCT ACATCTGCCA AGTGGAGCAC ACCAGCCTGG T ---C ...... ............... T ....... C ...............................
CCCCCAGCAG
ATAGTTCCGT CT C-T--
DPwla DPwlb DPw4
exon 4 CACCGTGGAG TGGAAGGCAC AGTCTGATTC TGCCCAGAGT AAGACATTGA CGGGAGCTGG ........................ = . . . . . . . . . . . . .G ..................... ................................... G ........................
DPwla DPwlb DPw4
exon GGGCTTCGTG CTGGGGCTCA TCATCTGTGG .................................... ....................................
DPwla DPwlb DPw4
exon 5 *** T GAAAGTTCAA CGAGGATCTG CATAAACAGG GTTCCTGACC TCACCGAAAA GACTAATGTG ...... .................... G ........... G ..... T .......... T .... ...................................... G ..... T .......... T ....
DPwla DPwlb DPw4
exon 6 CCTTAGAACA AGCATTTGCT GTGTTTTGTT AACACCTGGT ...... G-A ................. C .... G--T .... C ...... G-A ................. C .... G--T .... C
DPwla DPwlb DPw4
exon 6 TCCCAAGAGG ATACTGCTGC CAAGAAGTTG CTCTGAAGTC AGTTTCTATC --,A--TT ........ ( ) .......... - .................... ---A--TT ..........................................
DPwla DPwlb DPw4
TTGATTCAAA GCACTGTTTC TCTCACTGGG CCTCCAACCA TGTTCCCTTC ............................................................ ............................................................
DPwla DPwlb DPw4
CACAAATAAT
exon
CAAAACCCAA
exon CATAAGTGTT
4 AGTGGGACTC TTCATGCACA GGAGGAGCAA CA ...................... CA ......................
TCCAGGACAG ACCCTCAGCT ............. T---A-............. T---A--
240
300
48O
540
600
660
720
780
840
900
GTTCTGCTCT A ......... A .........
960
TTCTTAGCAC
1020
6
6 TGCTTTCCTT
T
.....................
D P w l a a n d D P w l b DPB1 alleles a r e still a p p a r e n t . A g a i n , D P w l b s e e m s m o r e closely r e l a t e d to o t h e r alleles t h a n to D P w l a . L i k e c o m p a r i s o n s o f alleles o f m a n y m a jor histocompatibility complex genes, a patchwork of
1061
Fig. 1. Nucleotide sequences of HLA-DPB1 cDNA clones, eDNA clones were isolated from the cell lines VAVY, RSH, BH, and DBB, as described (Sartoris et ah 1987). Numbers in the right margin indicate the position of the last nucleotide in the line, with the DPwla sequence as the reference. Dashes in the DPwl and DPw4 DPBt sequences indicate similarity and only substitutions are shown. A small deletion in the DPwlb sequence is indicated by 0. • denotes exon boundaries and *** indicates the termination codon
s u b s t i t u t i o n s b e t w e e n alleles a r e f o u n d a n d i s o l a t e d a m i n o acid o r n u c l e o t i d e c h a n g e s a r e u n u s u a l . A n a l y s e s for the T e n t h I n t e r n a t i o n a l H L A W o r k s h o p s h o w e d t h a t the cell line V A V Y e x p r e s s e s t h e D P A 1 1.1
348
J.S. Lee et al.: Sequence polymorphism of HLA-DP beta chains sequence
signal DPwla
MMVLQVSAA
DPw4a
]
first
DPwla DRwlb DPw2
RATPENYVYQ
DPw3 DPw4a DPw4b
GRQECYAFNG
-HLL
TQRFLERYIY
PRTVALTALL MVLLTSVVQG .....................
domain NREEYARFDS
DVGEFRAVTE
LGRPAAEYWN
.........................
FV ..................
DE ....
LF .........................
FV ..................
DE ....
.......... L ....................... ....... LF .........................
FV .................. F .........................
DED---
.......
FA ..................
DE ....
.......
-i
LF .........................
60
pII~7
[ DPwIa
SQKDILEEKR
DRwlb DPw2
...............
AVPDRVCRHN
YELDEAVTLQ
M .......
GGPM
RRVQpKVNVS ........
PSKKGPLQHH
NLLVCHVTDF
DPw3 DPw4a
...............
GGPM
........
R ........................
DPw4b
............... M ....... GGPM ....................................
........
R ........................ _ .......................
pIIfl7
M .......
second DPwla DRwlb DPw2 DPw3 DPw4a DPw4b
YPGSIQVRWF
LNGQEETAGV
VSTNLIRNGD
pIIfl7
................................ ................................ ................................ ................................ ................................ ................................
DPwla
SVTVEWKAQS
DRwlb DPw2 DPw3
P P P P P
[
DPw4a DPw4b pII$7
............ ............ ............ ............ ............
DSAQSKTLTG R R R R R
transmembrane AGGFVLGLII
.................... .................... .................... .................... ....................
P .................................
120
R ........................
........ E ...... M ....... GGPM ........ R ........................ .... L .......................................................
domain WTIQILVMLE F F F F F F
MTPQQGDVYI
................ T ................ T ........................... ................ T ................ T ...........................
region CGVGLFMHRR I I I I I
l
CQVEHTSLDS
180
.......... .......... .......... ....... M--
cytoplasmic
domain
SKKVQRGSA
229
.............. .............. .............. .............. ..............
I ........
GEKACKFNEDLHKQGS
polypeptide, while RSH expresses both DPA 1 1.1 and 1.2 (Knowles 1989), but neither DPA1 allele is associated with particular DPw types. It is intriguing that although cellular typing was unable to detect clearly the DPwlb allele, extra reactions were found for the cell line RSH (Mickelson et al. 1988). These extra reactions may be due to partial cross reactions of the DPw lb product with T-cell receptors recognizing the other DPB1 alleles to which the DPwlb allele is closely related. The DPw4 DPB1 cDNA clones we isolated from cell lines of different haplotypes (BH and DBB) are identical throughout the coding region and 3' untranslated region, and are identical to other DPw4 sequences that are available (Tonnelle et al. 1985, Kappes et al. 1984, Gorski et al. 1984, Gustafsson et al. 1984). Even though the DPw4a DPB1 sequence differs from DPw4b DPB1, only three replacements are found in the first domain, and one of them is conservative (Fig. 2; Lair et al. 1988). In this case, the first or third hypervariable regions in the first domain could be dominant in allospecific recognition of DPw4. Taken together, our findings show that cellular assays cannot accurately detect DP alleles. Future typing for HLA-DP should be oriented toward a better definition of these specificities, either using monoclonal antibodies or DNA-based typing with polymerase chain reaction (PCR) amplified DNA and specific oligonucleotides.
Fig. 2. Amino acid sequence of HLA-DPB1 alleles. Sequences for the alleles were obtained from the following sources: DPwla and DPwlb (this report), DPw2 (Gustafsson et al. 1987), DPw3 (Kappes et al. 1984), DPw4a (this report, identical to DPw4 in Figure 1), DPw4b (Lair et al. 1988), and pli/37 (Gustafsson et al. 1984). Numbering in the right margin indicates the position of each residue in the mature protein ( - 1 - 2 9 are the signal peptide), and vertical lines above residues indicate the boundaries of exons. As in Figure 1, dashes indicate identity, and only substitutions are shown
Acknowledgments. We thank Drs. B. Dupont, R. Knowles, N. Flornenberg, C. Hume, and L. Shookster for helpful discussions and advice, and P. Falldorf for technical assistance. This work was supported by grams from the Multiple Sclerosis Society (RG 1937-A-1) to J. S. Lee and the National Institutes of Health (AI-22960-01) to B. Dupont and J. S. Lee.
References Bell, J.I., Denney, D. Jr., Foster, L., Belt, T., Todd, J.A., and McDevitt, H. O. : Allelic variation in the DR subregion of the human major histocompatibility complex. Proc Natl Aead Sci USA 84: 6234-6238, 1987 Gorski, J., Rollini, P., Long, E., and Mach, B.: Molecular organization of the HLA-SB region of the human major histocompatibility complex and evidence for two SB beta-chain genes. Proc Natl Acad Sci USA 81: 3934-3938, 1984 Gustafsson, K., Emmoth, E., Widmark, E., Bohme, J., Peterson, P. A., and Rask, L.: Isolation of a cDNA clone coding for an SB betachain. Nature 309: 76-78, 1984 Gustafsson, K., Widmark, E., Jonsson, A.K., Servenius, B., Sachs, D.H., Larhammar, D., Rask, L., and Peterson, P.A.: Class II genes of the human major histocompatibility complex. Evolution of the DP region as deduced from nucleotide sequences of the four genes. J Biol Chem 262: 8778-8786, 1987 Kappes, D. J., Arnot, D., Okada, K., and Strominger, J. L.: Structure and polymorphism of the HLA class I1 SB light chain genes. EMBO J 3: 2985-2993, 1984 Knowles, R. W. : Assignment of HLA-class 1I ~ and/3 chain 2-D gel patterns for the Workshop reference panel of B lymphoblastoid cell
J.S. Lee et al. : Sequence polymorphism of HLA-DP beta chains lines. In B. Dupont (ed.): Immunobiology ofHLA Vol. I, Springer Verlag, New York, 1989 Korman, A. J., Boss, J. M., Spies, T., Sorrentino, R., Okada, K., and Strominger, J.L.: Genetic complexity and expression of human class II histocompatibility antigens. Immunol Rev 85: 45-86, 1985 Lair, B., Alber, C., Yu, W.-Y., Watts, R., Bahl, M., and Karr, R. W. : A newly characterized HLA-DP B-chain allele. Evidence for DP /3 heterogeneity within the DPw4 specificity. J Immunol 141: 1353-1357, 1988 Mickelson, E., Reinsmoen, N., Robbins, F.M., et al.: HLA-Dw and HLA-DP typing of the reference panel of B-lymphoblastoid cell lines. In B. Dupont (ed.): Immunobiology ofHLA, Vol. I, Springer Verlag, New York, 1989 Mullis, K.B. and Faloona, F.A.: Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155: 335-350, 1987 Nomenclature Committee on Leukocyte Antigens. Nomenclature for factors of the HLA system, 1987. Immunogenetics 28: 391-398, 1988
349 Sanger, F., Nicklen, S., and Coulson, A. R. : DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467, 1977 Sartoris, S., Cohen, E. B., and Lee, J. S. : A rapid and improved method for generating cDNA libraries in plasmid and phage lambda vectors. Gene 56: 301-307, 1987 Simons, M. J., Wheeler, R., Lalouel, J-M., and Dupont, B. : Restriction fragment length polymorphism of HLA genes: Summary of the 10th International Workshop Southern Blot Analysis. In B. Dupont (ed.): lmmunobiology ofHLA, Vol. I, Springer Verlag, New York, 1989 Tonnelle, C., DeMars, R., and Long, E. O.: DOt~: a new/3 chain gene in HLA-D with a distinct regulation of expression. EMBO Y 4: 2839-2847, 1985 Wank, R. and Schendel, D. J.: Genetic analysis of HLA-D region products defined by PLT. In E. D. Albert, M. P. Baur, and W. R. Mayr (eds.): Histocompatibility Testing 1984, pp. 289-299, SpringerVerlag, Berlin, 1984 Received October 10, 1988; revised version received January 12, 1989
