Immunogenetics 38: 141-145, 1993 //H///////o- genedcs

© Springer-Verlag 1993

Characterization of the rhesus macaque (Macaca mulatta) equivalent of HLA-F Nel Otting, Ronald E. Bontrop

Department of Chronic and Infectious Diseases, ITRI-TNO, P.O. Box 5815, 2280 HV Rijswijk, The Netherlands

Received December 28, 1992

Abstract. Nucleotide sequence analysis of rhesus ma- caque major histocompatibility complex class I cDNAs allowed the identification of the orthologue of HLA-F, designated Mamu-F. Comparison of Mamu-F with ear- lier published human and chimpanzee orthologues demonstrated that these sequences share a high degree of similarity, both at the nucleotide and amino acid level, whereas a New World monkey (cotton-top ta- marin) equivalent is more distantly related. Exon 7, encoding one of the cytoplasmatic domains, is absent for all primate Mhc-F cDNA sequences analyzed so far. In contrast to the human, chimpanzee, and rhesus ma- caque equivalents, the cotton-top tamarin Saoe-F gene seems to have accumulated far more nonsynomynous than synonymous differences.

Introduction

The major histocompatibility complex (Mhc) can be divided into two major segments, encoding transmem- brahe structures, designated the class I and II regions. In humans, the class I region contains the loci for the classical transplantation antigens; these are HLA-A, -B, and -C, which are expressed on a wide diversity of tissues. These class I molecules present foreign pep- tides from intracellular origin to cytotoxic T cells, which subsequently may lyse the target cell. To execute this function, Mhc molecules are equipped with a pep- tide binding site (Bjorkman et al. 1987). Transplanta-

The nucleotide sequence data reported in this paper have been sub- mitted to the Genbank nucleotide sequence database and have been assigned the accession number Z 21819.

Correspondence to: R. E. Bontrop.

tion antigens display abundant polymorphism in the population (Parham et al. 1989), and as a consequence different alleles may select distinct peptides for T-cell activation. The extreme degree of allelic diversity is maintained by overdominant selection (Hughes and Nei 1988). Equivalents of HLA-A,-B, or-C loci have been detected in chimpanzees (Balner et al. 1974; Lawlor et al. 1988; Mayer et al. 1988); gorillas (Lawlor et al. 1992), orangutans, gibbons (Lawlor et al. 1990; Chen et al. 1992), and rhesus macaques (van Vreeswijk et al. 1977; Miller et al. 1991).

Apart from these classical transplantation antigens, the HLA class I region contains several nonclassical genes named HLA-E, -F, -G, and -H (Koller et al. 1989; Orr 1989; WHO 1992). At least one of them, HLA-H, is clearly a pseudogene (Orr 1989; Zemmour et al. 1990). As was found for the classical transplantation antigens, the HLA-E, -F, and -G gene products complex with ~32-microglobulin. In contrast to the classical transplan- tation loci, however, the nonclassical HLA or H2 genes are mono- or oligopolymorphic, show a limited tissue distribution, and may display specialized functions (Wei and On" 1990; Hedrick 1992). Equivalents of the HLA-E locus have been detected in the orangutan (Watkins et al. 1992), whereas ortholoques of the HLA-F locus have been identified in the chimpanzee (Lawlor et al. 1988, 1990) and cotton-top tamarin, a New World primate species (Watkins et al. 1990). Remarkably, at least 11 alleles have been identified for the Saoe-G locus in the cotton-top tamarin (Watkins et al. 1990, 1991a, b).

Resistance to collagen type-II-induced arthritis in rhesus monkeys is controlled by a particular Mamu-A allele, or alternatively by a closely linked gene (Bakker et al. 1992). To gain insight into the mechanisms under- lying this association, we began to analyze the class I region. In the process of sequencing Mamu class I cDNAs an ortholoque of the HLA-F locus was identi-

142 N. Otting and R. E. Bontrop: HLA-F equivalent in macaque

fled. The nucleotide and deduced amino acid sequence of the Mamu-F gene is reported in this communication and compared to available homologues of other primate species.

Materials and methods

Cells. Transformed B cells of rhesus macaque KM (Mamu- A26/B23) were grown in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum, glutamine, penicillin, and strep- tomycin.

Preparation and amplification of cDNA. RNA was isolated accord- ing to standard procedures, whereas the commercially available Riboclone kit (Promega, Madison, WI) was used to prepare cDNA. The protocol and primers used for amplification of Mhc class I cDNAs have been described previously (Ennis et al. 1990).

Subcloning and sequencing. Polymerase chain reaction products were digested with the restriction enzymes Sal I and Hin dIII and cloned into the M13 derivatives tgl30 and tgl31 (Kieny et al. 1983). Inserts were sequenced by the dideoxy chain termination method (Sanger et al. 1977) using ~SSa-a-thio-ATP and modified T7 DNA polymerase (Promega). The reported Mamu-F sequence represents the consensus of five independent clones.

Results and discussion

As can be seen, the nucleotide sequence of the Mamu-F cDNA has been aligned with its human (HLA-F), chim- panzee (Patr-F), and cotton-top tamarin (Saoe-F) equivalents (Fig. 1). Apart from diagnostic nucleotide substitutions, some features distinguish the Mhc-F locus from other types of Mhc class I genes. For exam- ple, the HLA-F nucleotide sequence, originally obtain- ed from genomic DNA, was found to possess a mutated 3' splice site in intron 6, resulting in the fact that the corresponding messenger lacks exon 7 (Orr 1989). This phenomenon seems to be more or less conserved in primates, since cDNA sequences obtained from chim- panzees (Lawlor et al. 1990), rhesus macaques (this communication), and tamarins (Watkins et al. 1991a, 1993) also lack exon 7 (Fig. 1). ff the absence of exon 7 is caused by one single genetic event, then the mutation of the 3' splice site in question is likely to have taken place prior to speciation of hominoids, Old-, and New World monkeys, starting about 35 million years ago (Pilbeam 1984).

It is noted that the Mhc-F locus gene products have a somewhat shorter leader peptide than most other Mhc class I sequences due to a mutation affecting the first ATG initiation codon. As a consequence, an ATG ini- tiation codon lying more upsteam is being employed (Fig. 1). Such observations were not only done for rhe- sus macaques but also in humans (Orr 1989) and chim- panzees (Lawlor et al. 1990), whereas for the tamarin (Watkins et al. 1993) this type of information is not

available, since the corresponding segment of exon 1 has not been sequenced (Fig. 1).

The rhesus macaque and tamarin sequences show a 6-nucleotide insertion in exon 2, which genetically seems to be unstable, since it differs between these two species (Fig. 1). The/-/LA- and Patr-F genes lack these 6 extra nucleotides (Fig. 1). It seems conceivable that this particular insert originates from one primordial event affecting an ancestral form of the Mhc-F gene. Since both the Old- and New World monkey repre- sentatives share the insert, the original integration must predate their speciation and must have taken place more than 35 million years ago. If this scenario is true, than this insert must have been lost somewhere along hom- inoid speciation.

The deduced amino acid sequences of the various primate Mhc-F genes are depicted in Figure 2. Differ- ences between the Mhc-F genes of different species are mainly explained by point mutations, Some of these mutations result in amino acid replacements, whereas others are silent (Figs. 1, 2). The degree of similarity of various sets of Mhc-F amino acid sequences has been calculated (Table 1). As can be seen, the HLA- and Patr-F sequences share a degree of 98.4% similarity. Of the 17 nucleotide differences only six resulted in amino acid replacements (Lawlor et al. 1991). The Mamu-F sequence shares a degree of similarity of about 94% with its hominoid equivalents. For this com- parison, of 54 differences 35 (human) or 34 (chim- panzee) turned out to be of a synonymous character. This comparison shows that the structure of the HLA-, Patr-F, and Mamu-F sequences has been maintained over long evolutionary spans of time. No evidence could be found that these genes have mutations that would render them pseudogenes. In this light it is pos- sible that there may be a biological role for the corre- sponding gene products. For the New World monkey sequence a disparate situation is observed. Comparison with Hominoid and Old World monkey ortholoques demonstrates that degrees of similarity reach levels of about 8 1 -8 2 % (Table 1). As expected, in these cases more non-synomynous than synonymous differences are observed. This may indicate that the Saoe-F sequence has diversified to a considerable extent and may be less subject to constraint than its homoloques. In the extreme situation the Saoe-F gene is, or may be, on the way to become a pseudogene. This situation is plausible, as is reflected by the nucleotide composi- tion of exon 5 encoding the transmembrane region (Figs. 1, 2). Exon 5 is affected by at least one deletion and a high number of non-synonymous differences (Figs. 1, 2). Possibly some of these alterations influ- ence the ability of the tamarin gene product to stay in a membrane-bound configuration. This is in agreement with the observation that transfection of the Saoe-F gene in COS cells did not result in expression of the corresponding gene product (Watkins et al. 1991).

N. Otting and R. E. Bontrop: H L A - F equivalent in macaque 143

l0 20 30 40 50 60 70

Exon 1 i I I I I consensus ATGCGGGTCATGGCGCCCCGAACCCTCC CCTGCTGCTCTCGGGGGCCCTGGCCCTGACCGAGACCTGGGCCG

HLA-F G-TG .................. G .................. a ................... T t ..... g

PBtz-F ......... G ....... T ---a ...................... t ..... a-

Mamu-F G-TO ............................. G ....... a ............................. g-

Saoe-F ................ T .......... A . . . . . . . . . . . . g

10 20 30 40 50 60 70 80 90

Exon 2 ] / I I I i r consensus GCTCCCAC?CCATGAGGTATTTCTACACCTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCC GCTTCATCGCAGTGGGCTACGTGGACGACA

HLA-F ........... T . . . . . . . . AG .... G-T -g ........ g . . . . . . . . . . . . . A ...... c .... AG ..... a .......

Patr-F ........... T .......... AG -G-T ..... g . . . . . . . . . . . . . @-A ........... AG ..... a .......

Mamu-F ........... T--c ....... tAG---tG-T ..... g ........... a . . . . . t-AGTAZA g ~ ..... g-- AG . . . . . . . .

Saoe-F ........... T ........... AGT--LG--A .......... t ......... GAT~CA-g-A .... G .............. t ....

100 110 120 130 140 150 160 170 180

I ] [ I I ] I

consensus CGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGTCCGAGGATGGAGCCG~GGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGG

HLA-F .... a --C . . . . . . . . . . . . . . . . T ........ A ....... G-G ..... a ......... C .......

Patr-F ........ C ........................ T ............... h .... A ....... G-G ..... a ......... C .......

Mamu-F ........ C ........................ T ................. a .... G-G a ..... C ....

Sa~e-F ---T . . . . . . . a . . . . . . . . . . . . . . . A-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-G ......... A ...............

190 200 210 220 230 240 250 260 270

I I ! L I I l consensus A~CGGGAGACACAGAACTTC/~AGGCCCACGCACAGACTGACCGAGAGAACCTGCGGAACCTGCGCGGCTA~TA~AACCAGAGCGAGGCCG

HLA-F GT -ACC---GG-T--GC ....... A .................. T-GC .... a ........ T-C--CG .................. t-

Patr-P -Ga--ACC---GG-T--GCG ...... A .................. T-GC .... a ......... T C--CG ...................

Mamu-F -G---ACC---GG-T--GC ....... A ...... G . . . . . . T-GC .... a- -G .... Y-CT-CG ....................

Saoe-F -GGACC ...... G-GT G ........ A . . . . . . . . . . . . . T---A ........ A .... T-C .......... t .......... G--

i0 20 30 40 50 60 70 80 90

E x o n 3 I I I f I I 1 consensus GGT~TCACA~CTC~AGAGGA~GTATGG~TG~GACGTGGGG~GGACGGG~G~TC~T~G~GGGTATGA~AGTACG~CTAcGA~GGCA

HLA-F ................. G-A---A .......... A ....... c ..... a ................ C ..... C .... g ..........

Patr-F ................. G-A---A . . . . . . . . . A . . . . . . c ..... a ........ t . . . . . . . . C - C ---g- - -

Mamu-F . . . . . . . . G A --A C .... A ...... c ..... a ................. C ..... C ..............

Sa~e-F ................. G-A-C-A .......... t ..... a--c--t--a---T .......... a---C ..... C ..............

i00 110 120 130 140 150 160 170 180

I I I I [ I I Consensus AGGATTACATCGCCCTGAA•GAGGACCTG•GCTCCTGGACCGCGGCGGA•ACGGCGG•TCAGATCACCCAGCGCAAGTGGGAGGCGGCCC

HLA-F ........... T .................................... c-T ............. TTC-AT .... a AGG

Patr-F ........... T ....................................... c-T . . . . . . . . . . . TTC-AT a-AGG

Mamu-F ........... T ............................... c .......... TA--- G ............. TTC-AT ..... a-ACG

gaoe-F c - -T . . . . . . . . . . . . . . . . . . . . . a ..... a . . . . . . . . . . . . . . . . . . . . . T AT T-a-AGA

190 200 210 220 230 240 250 260 270

I I I I I I i I consensus GTGTGGCGGAGCAGCTGAGAGCCTACCTGGAGGGCACGTGCGTGGAGTGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCGCGG

HLA-F AATAT--a---G--T-C--gA .............. GA .... C ...... T ............. t ....... t .............. a ........ a-

Patr-E AATAT--a---G--T-C--gA . . . . . . . . . . GA .... C ...... T ............. t . . . . . . . . . a ........ a-

Mamu-F AATAT--a---G--T-C--gA ........... GA C ..... T . . . . . . . . . . . . . . . . . . . . . . a ....... a-

Saoe-F AATAT--a---G~-T-C--cA ........ A ..... GG ................. A ....................................... t-

I0 20 30 40 50 60 70 80 90

E x o n 4 [ I I I I I I i consensus ACCCCCCAAAGA•ACATCTGAC•CA•CACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTAC•CTGAGGAGA

HLA-F -t--t- - G- -c--tG ...................................................................

Patr-F -t--t ...... G .... cA-TG .....................................................................

Namu-F -t--t ...... G .... c--tG ........... G ......... G .......................................... c ....

Saoe-F TT--t ...... G .... c--tG . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . .

I00 ii0 120 130 140 150 160 170 180

I I I I I I I I c~nsensus T•ACACTGACCTGGcAGcGGGATGGGGAGGACCAAACTCA•GACACGGAGCTTGTGGAGACCAGGCCAGCAGGAGATGGAACCTTCCAGA

HLA-F .... g ......................... A- g--c ........ a .................... t ..... g ................

Patr-F .... g .......................... A--g--c ........ a . . . . . . . . . . . . . . . . t ..... g ....... A . . . . . .

Mam~-F . . . . . . . . . . . . . . c -- -A -g--c ............................. t ..... g ................

Saoe-F -t-TG ............ a ....... a ..... t -g--c ........ a--a ............... t --g . . . . . . . . . . . . .

Fig. 1. Nucleotide sequence align- 190 2~0 at0 220 230 240 250 260 270

I I I , I r i I merit of human ~u~,knL~), chim- consensus AGTGGG~AG~TGTGGTGGTGCCTT~TGGAGAAGAGCAGAGATA~ACATG~ATGTG~AG~ATGAG~GG~TGCCGAAGCC~CT~ACC~TGAGATGGG panzee ( P a t r ) , rhesus macaque HLA-F ..... c . . . . . . . . . . . . . . . . g--a .......................... c ........... cC ......... T ...........

Pat~-F ....... t ..................... ~--~ ........... ~ . . . . . . . . . . . . . ~ ........... cC ..................... ( M a m u ) , and cotton-top tam.in Mamu-F ......................... c ..... q . . . . . . . . . g--- a ..... a - - cC . . . . . . . .

s~o~-~ . . . . . . . . . . . ~ . . . . . . ~ - - g . . . . . . o . . . . . . . o . . . . . . . . . . . . . . o . . . . . . . . . . . ~G--Z . . . . . . . . . . . . . . . . . . ~ "[Jaoe) M h c - F ~ " "aneies. The -top l i n e

10 20 30 40 50 60 70 B0 90

E x o n 5 ] I I I I I I I consensus AGCCGT•TTC•CAG•CCACCAT•CCCAT•GTGGGCAT•GTTGCTGGCCTGG•TGTCCTTGCAGT•CTTGTGGT•A•TGGAG•TG7GGTCG

HLA-F ---A .... C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t T ...... G ***C-- C . . . . . . . .

Parr F ---A- C ................................... t-T ........ G--***C ........ C ..............

Mamu-F --T ....... t ................. t ............................. a .... *** ....... t-C ..............

Saoe-F .... a ............... G---T .... A ........ AC ...... tG---T . . . . G--***C . . . . . C . . . . . . . ****

100 110 120

consensus CTGCTGTGATGTGTAGGAGGAAGAGCTCAG

HLA-F ....... g .... A ...........

Patr-F ............. g .... A ...........

Mamu-F . . . . . . . a ..... g -- A . . . . . . . . . .

Saoe-F ******** ..... g .... A .... T TGTTGGGAAGGG

I0 2Q 30 1 I0 20 30 q0 50 1

Exon 6 S I I I I / ; I I I consensus GTGGAAAAGGAGGAAGCT~TCTCAGGCTGCGTGCAG~GACAGTG~CCAGGGCTCTG~TGTGTCT~TCACAGC~TGTAAA~TGTGA

HLA-F a-A .... CA ...................... aGT--CT ................. GG . . . . . . . . . . . i~ . . . . . .

Patr-F a-A .... CA . . . . . . . . . . . . . . . . . ************************************************** .....

Mamu-F a-A .... CA ...................... ************************************************** .....

Saoe-F a-A .... CA ...................... ************************************************** .....

represents a consensus M h c class I sequence. Identity to this sequence is indicated by a d a s h ,

non-synonymous and synony- mous substitutions by c a p i t a l s a n d

l o w e r - c a s e characters, respec- tively, whereas gaps to the con- sensus are depicted by an a s t e r i s k .

The ATG initiation codon has been u n d e r l i n e d . The HLA-F (Orr 1989), P o t r - F (Lawlor et al. 1990), and S a o e - F (Watkins et al. 1993) sequences were taken from the literature.

144 N. Otting and R. E. Bontrop: HLA-F equivalent in macaque

Leadez peptide consensus HLA-F

~atr-F

Mamu-F Saoe-F

Alpha 1

co.senNas

HLA-F

Patr-F

Mamu-F Saoe-F

Alpha 2

co~sens~ HLA-F

Patr-F

Mamu-F Saoe-F

Alpha 3

co~8e~s~s

HLA-F

Patr-F

Mamu-F Saoe-F

-i0 [

MAPI~TL LLL L S GAJ=AL TE TWA

.... S ............ D---

.... S .... F ...........

........ V ............

..... L---T ......

I(] 20 30 40 50 60 70 80 90

l J 1 l ] b l I l GSHEMRYFYTSVS KPGRGEP P~ [AVG YVDDTQFV~DSDAAS P BMEPRAgW I EQEGPEYNDRE TQN FKAHAQT DRENLKNLRGYYNQS EA

.... L---S-A .......... Y- --E ...... L--- I ...... E V .... Q--EWT-GYA--N ..... VA .... LP~ --

.... L---S-A .......... Y---E ....... L ....... i ...... E--V ..... Q--E -T-GYA--N ..... VA .... LRR .....

.... L---S-A ......... QY-Y---E ....... L ....... I . . . . . . V --Q=-E-T GYA--N-R---VA -K-LLR ......

-L---S-AM ..... RY-Y-G ....... L: . . . . DP ......... V---R .... EDQ-RW--N ..... VK--K-LR ...... G

i00 ii0 120 130 140 15D 160 170 180

J I 1 I I I I l I GSHTLQRMYGCDVGP DGP~L LRG YD Q YAY DGKD Y I ALNZ DLRSWTAAD TAAQ I T Q P~KWEAARVAEQLRAYLE GTCVEWLK.~VLENGKE TL QF<A

...... G-N- --M .......... ~-H ........ ~ ............. V ...... FY--KEY--EF-T .... E-L-L ............

...... G-N---M .......... B-H ........ s ............ v- -FY--EEY- EF T --E-L L ........

-G-N- M ........ H-H ........ S ........... V-R--- FY EEY--EF-T .... R-L-L ...............

...... GTN ......... F .... H-H ....... S .................. MY-SEKY-- EFST--K-G .... H . . . . . .

190 2gO 210 220 230 2~0 250 260 270 q

DPPKTHV~HH~ I S DHNATL RCWALGFYP AE I TLTWQRD GED QTQD TE LVE TFtP AGD G T FQKWAA~ArVP $ GE EQRY TC HVQHEGLP Kp LT LRW

.... A--A ................................ E ........................................ Q 1 - --

. . . . A - I A . . . . . . . . . . . . . . . . . . . . . E . . . . . . . N . . . . . . . . . . . . . . . . . . . . . Q -

.... A--A-- -V- -R ......................... E ............................................ Q ......

V---A--A ......... I ............. M ................................... L . . . . . . . S --

280 290 3QO 310

Transmembrane J J I I consensus EPSSQPTIPIVGIVAGLAVLA%rLWIGAWAAVMCBRKSS

HLA P -Q P ....... V--~A*--T .......... K---

Patr-P -Q-P ............. V--GA*--T .......... K---

Mamu-F -S .................... * T ....... K

Saoe-F ....... VL-M--T--W--GA*--T---****---K-IWLGR

Cygoplasma~ic domains

320 330 340 l [

consensua GGKGGSYSQ~ CSDSAQGSDVSLTACK V

HLA-F DRNR ....... VT ...... G ..... N- -

Batr-F DRNK ....... ***************~

Mamu-F DRNR ....... ***************~ -

5aoe-F D~NR ....... ****************

Fig. 2. Alignment of deduced ami- no acid sequences of various pri- mate Mhc-F sequences.

Table 1. Percentages of amino acid sequence similarity observed between various sets of primate Mhc-F sequences.

P atr Mamu Saoe

HLA 98.4% 94.5% 82% Parr - 94.2% 81.7% Mamu - - 81.0%

In conclusion, these data show that the Mhc-F gene has been conserved in hominoid and Old World primate species, whereas its equivalent diversified considerably in a New World primate species and possibly may constitute a pseudogene in tamarins.

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