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A Novel Family of Human Leukocyte Antigen Class II Receptors May Have Its Origin in Archaic Human Species*

Open AccessPublished:November 08, 2013DOI:https://doi.org/10.1074/jbc.M113.515767
      HLA class II α and β chains form receptors for antigen presentation to CD4+ T cells. Numerous pairings of class II α and β subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched αβ heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study we investigated assembly of isotype-mixed HLA class II α and β heterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed α and β subunits was dependent on the groove binding section of the invariant chain (Ii). By mutation of polymorphic DPβ sequences, we identified two motifs, Lys-69 and GGPM-(84–87), that are engaged in Ii-dependent assembly of DPβ with DRα. We identified five members of a family of DPβ chains containing Lys-69 and GGPM 84–87, which assemble with DRα. The Lys/GGPM motif is present in the DPβ sequence of the Neanderthal genome, and this ancient sequence is related to the human allele DPB1*0401. By site-directed mutagenesis, we inspected Neanderthal amino acid residues that differ from the DPB1*0401 allele and aimed to determine whether matched heterodimers are formed by assembly of DPβ mutants with DRα. Because the *0401 allele is rare in the sub-Saharan population but frequent in the European population, it may have arisen in modern humans by admixture with Neanderthals in Europe.

      Introduction

      Major histocompatibility class II (MHCII) molecules present antigenic peptides to T cells. In humans the majority of MHCII molecules are composed of isotype-matched α and β subunits from HLA-DR, DP, or DQ. Assembly of α and β chains, which takes place after biosynthesis in the ER,
      The abbreviations used are:
      ER
      endoplasmic reticulum
      aa
      amino acid
      Endo H
      endoglycosidase H
      PNGase F
      peptide N-glycosidase F
      MHCII
      major histocompatibility complex class II
      Ii
      invariant chain
      HLA
      human leukocyte antigen
      contig
      group of overlapping clone.
      may involve the initial formation of mixed isotype complexes (
      • Koch N.
      • McLellan A.D.
      • Neumann J.
      A revised model for invariant chain-mediated assembly of major histocompatibility complex class II assembly and isotype pairing.
      ). Why isotype-matched assembly of MHCII subunits is more efficient than inter-isotype αβ pairing is still unclear. The primary sequence of MHCII α and β chains may endow properties that ensure isotype-specific interactions. In addition to isotypic differences, the polymorphism of the MHCII genes modifies the ability of the encoded α and β subunits to acquire the appropriate conformation (
      • Bikoff E.K.
      • Germain R.N.
      • Robertson E.J.
      Allelic differences affecting invariant chain dependency of MHC class II subunit assembly.
      ,
      • Lechler R.I.
      • Sant A.J.
      • Braunstein N.S.
      • Sekaly R.
      • Long E.
      • Germain R.N.
      Cell surface expression of hybrid murine/human MHC class II β α dimers. Key influence of residues in the amino-terminal portion of the β1 domain.
      ). A quality control of the assembled MHCII heterodimers in the ER precedes subsequent adaption of the polypeptides to the class II processing pathway. In 1992 Anderson and Miller (
      • Anderson M.S.
      • Miller J.
      Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules.
      ) showed that invariant chain (Ii) is a chaperone for MHCII molecules that affects the conformation of the αβ heterodimer. By transfection of class II cDNAs, it was demonstrated that some MHCII allotypes assemble independent of Ii, whereas for other α and β combinations, co-expression of Ii is required for pairing and subsequent cell surface expression (
      • Layet C.
      • Germain R.N.
      Invariant chain promotes egress of poorly expressed, haplotype-mismatched class II major histocompatibility complex AαAβ dimers from the endoplasmic reticulum/cis-Golgi compartment.
      ). Intracellular transport of haplotype-mixed (α and β chains encoded on different parental chromosomes) MHCII heterodimers is promoted by associated Ii chain (
      • Layet C.
      • Germain R.N.
      Invariant chain promotes egress of poorly expressed, haplotype-mismatched class II major histocompatibility complex AαAβ dimers from the endoplasmic reticulum/cis-Golgi compartment.
      ). In concert with the chaperone role of Ii, Ii gene-deficient mice showed a strongly reduced expression of MHCII on the plasma membrane of their antigen-presenting cell, and again the level of surface exposure was shaped by the MHCII haplotype (
      • Bikoff E.K.
      • Huang L.Y.
      • Episkopou V.
      • van Meerwijk J.
      • Germain R.N.
      • Robertson E.J.
      Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression.
      ). Recent data suggest that Ii is a specialized chaperone that facilitates assembly of matched MHCII heterodimers and segregation of disfavored combinations (
      • Neumann J.
      • Koch N.
      A novel domain on HLA-DRβ chain regulates the chaperon role of invariant chain.
      ). At the atomic level, it is not entirely clear which interactions of Ii with MHCII residues regulate folding of the αβ heterodimer.
      As intermediates of class II assembly, the various α and β subunits form macromolecular complexes in the ER (
      • Koch N.
      • Zacharias M.
      • König A.
      • Temme S.
      • Neumann J.
      • Springer S.
      Stoichiometry of HLA Class II-invariant chain oligomers.
      ). Subsequent subunit assembly is a requirement for disaggregation. In principle, β subunits from any class II locus, expressed in a heterozygotic antigen-presenting cell, can interact with DRα, which is monomorphic, or with any of the two allotypes each of DPα and DQα. Because of the many combinations that are statistically possible, a random interaction of nascent α and β chains in the ER would mean that initially a substantial number of isotype-mismatched αβ pairs is formed. Dissociation of mismatched and selection of matched αβ heterodimers is required for production of functional class II peptide receptors (
      • Koch N.
      • McLellan A.D.
      • Neumann J.
      A revised model for invariant chain-mediated assembly of major histocompatibility complex class II assembly and isotype pairing.
      ). In mice and humans, respectively, a combination of certain IA with IE or of DR with DP or DQ subunits to isotype-mixed heterodimers was demonstrated in transfected fibroblast cells and in EBV-transformed B cells (
      • Malissen B.
      • Shastri N.
      • Pierres M.
      • Hood L.
      Cotransfer of the Edα and Adβ genes into L cells results in the surface expression of a functional mixed-isotype Ia molecule.
      ,
      • Germain R.N.
      • Quill H.
      Unexpected expression of a unique mixed-isotype class II MHC molecule by transfected L-cells.
      ,
      • Lotteau V.
      • Teyton L.
      • Burroughs D.
      • Charron D.
      A novel HLA class II molecule (DRα-DQβ) created by mismatched isotype pairing.
      ). Some inter-isotype paired IA/IE heterodimers were functional in mixed lymphocyte reaction and by antigen presentation assays (
      • Spencer J.S.
      • Kubo R.T.
      Mixed isotype Class II antigen expression.
      ,
      • Ruberti G.
      • Sellins K.S.
      • Hill C.M.
      • Germain R.N.
      • Fathman C.G.
      • Livingstone A.
      Presentation of antigen by mixed isotype class II molecules in normal H-2d mice.
      ). In addition, mice that only expressed mixed haplotype class II molecules were able to mediate selection of functional CD4+ T cells (
      • Silk J.D.
      • Schoendorf D.
      • Bartok I.
      • Chai J.G.
      • Gray D.
      • Simpson E.
      • Dyson J.
      Mixed-haplotype MHC class II molecules select functional CD4+ T cells.
      ). Structural constraints for the formation of intra- or inter-isotype pairing of MHCII heterodimers have not yet emerged.
      Meanwhile, sequences of several hundred DR, DQ, and DP alleles have been identified, and the structures of the corresponding heterodimers can be devised based on the published x-ray crystal structure of MHCII heterodimers (EMBL-EBI; Refs.
      • Stern L.J.
      • Brown J.H.
      • Jardetzky T.S.
      • Gorga J.C.
      • Urban R.G.
      • Strominger J.L.
      • Wiley D.C.
      Crystal structure of the human class II MHC protein Hla-DR1 complexes with an influenza virus peptide.
      and
      • Ghosh P.
      • Amaya M.
      • Mellins E.
      • Wiley D.C.
      The structure of an intermediate in class II MHC maturation. CLIP bound to HLA-DR3.
      ). The amino acid (aa) residues that form the contact sites between α and β chains in the polymorphic MHCII α1β1 domain are assumed to regulate intracellular assembly of the heterodimers (
      • Braunstein N.S.
      • Germain R.N.
      Allele-specific control of Ii molecule surface expression and conformation. Implications for a general model of Ii structure-function relationship.
      ). Conserved residues in the β2 domain underneath the peptide binding groove are also important for determining MHCII αβ chain pairing (
      • Murthy V.L.
      • Stern L.J.
      The class II MHC protein HLA-DR1 in complex with an endogenous peptide. Implications for the structural basis of the specificity of peptide binding.
      ). We set out to investigate the assembly of HLA-DR/DP isotype-mixed α and β subunits with Ii.

      DISCUSSION

      The αβ heterodimer composed of DRα and DPB1*0401 is an example of a functional isotype-mixed αβ heterodimer. To unravel the impact of polymorphic residues influencing class II α and β subunit assembly, we systematically determined the interaction of polymorphic residues in DPβ allotypes with DRα. These experiments were conducted in the presence or absence of the chaperone Ii. Ii controls carbohydrate maturation of the α-β heterodimer, and we show here that the DRA.DPB1*0401 molecules acquire SDS resistance (indicating peptide binding) and are expressed on the cell membrane. We demonstrate that allotypic differences in DPβ determine the assembly of isotype-mixed DRαDPβ heterodimers with Ii. Three additional DPβ allotypes associate with the DRα glycoprotein in the presence of Ii and form complexes, but their conformations did not lead to appropriate carbohydrate maturation. One complex composed of DRα and DPB1*0201 was exported from the ER and reached the cell surface, but even in the presence of Ii it did not acquire the conformation required for a normal carbohydrate modification. Two further isotype-mixed αβ combinations, which were co-expressed with Ii, formed complexes containing Ii but did not assemble as heterodimers. An imperfect conformation of αβ heterodimers yields aggregation rather than a rescue of conformation preceding binding of peptides (
      • Germain R.N.
      • Rinker Jr., A.G.
      Peptide binding inhibits protein aggregation of invariant-chain free class II dimers and promotes surface expression of occupied molecules.
      ). Lack of surface transport has been ascribed to a mechanism for limiting the expression of inappropriate paired murine MHCII subunits (
      • Sant A.J.
      • Hendrix L.R.
      • Coligan J.E.
      • Maloy W.L.
      • Germain R.N.
      Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex α/β chains.
      ).
      Ii promotes optimal MHCII assembly that results after release of Ii in a receptor susceptible for peptide binding. When the structure of different peptide-MHCII complexes was compared, the conformations of side chains of MHCII residues contacting the peptide were highly conserved (
      • Murthy V.L.
      • Stern L.J.
      The class II MHC protein HLA-DR1 in complex with an endogenous peptide. Implications for the structural basis of the specificity of peptide binding.
      ). Possibly, Ii converts the αβ heterodimer into a unique conformation that is sustained after binding of the peptide. Consistently, various peptides confer only minimal rearrangement of MHCII residues (
      • Murthy V.L.
      • Stern L.J.
      The class II MHC protein HLA-DR1 in complex with an endogenous peptide. Implications for the structural basis of the specificity of peptide binding.
      ). Upon release of the Ii-derived fragment CLIP from the MHCII cleft, the heterodimer becomes more flexible. This was previously demonstrated by a molecular dynamic simulation study (
      • Yaneva R.
      • Springer S.
      • Zacharias M.
      Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states. A molecular dynamics simulation study.
      ).
      The groove binding segment of Ii (CLIP) appears to be critical to shape the conformation of the MHCII peptide receptor. In recent years recombinant Ii chains have been described in which the CLIP sequence was replaced by the sequence of antigenic peptides (
      • Sanderson S.
      • Frauwirth K.
      • Shastri N.
      Expression of endogenous peptide-major histocompatibility complex class II complexes derived from invariant chain-antigen fusion proteins.
      ,
      • Koch N.
      • van Driel I.R.
      • Gleeson P.A.
      Highjacking a chaperone. Manipulation of the MHC class II presentation pathway.
      ). During assembly of α and β subunits in the ER, the Ii fusion protein is embedded in the MHCII cleft, and the MHCII molecule presents the antigenic peptide on the cell surface for activation of CD4+ T cells. One can envisage that some Ii-antigen fusion proteins may select unusual isotype-mismatched MHCII heterodimers to assemble on the scaffold of the antigenic peptide in the ER.
      The structural requirements for isotype-mismatched MHCII subunit assembly have been explored by species mixed combinations of DRα with mouse IAk β chain and monitoring of cell surface expression (
      • Norcross M.A.
      • Raghupathy R.
      • Strominger J.
      • Germain R.N.
      Transfected human B lymphoblastoid cells express the mouse Adβ chain in association with DRα.
      ). Construction of chimeric molecules of two HLA isotypes or interspecies fusion proteins indicate that the β1 domain impacts on isotype matched assembly of MHCII heterodimers (
      • Karp D.R.
      • Teletski C.L.
      • Jaraquemada D.
      • Maloy W.L.
      • Coligan J.E.
      • Long E.O.
      Structural requirements for pairing of α and β chains in HLA-DR and HLA-DP molecules.
      ). Cell surface expression of Aαkd heterodimers was induced by mutation of polymorphic residues at the position 76 and 86 of Aβk chain (
      • Buerstedde J.-M.
      • Pease L.R.
      • Nilson A.E.
      • Bell M.P.
      • Chase C.
      • Buerstedde G.
      • McKean D.J.
      Regulation of murine MHC class II molecule expression.
      ). Interaction of Ii with the MHCII groove is an essential requirement for assembly of some α and β allotypes. Amino acid residues from both the α and the β chains form the P1 pocket. This pocket has an important role for interaction of the MHCII subunits, for contact of the upper-lower domains of DR1, and for binding of Ii (
      • Sato A.K.
      • Zarutskie J.A.
      • Rushe M.M.
      • Lomakin A.
      • Natarajan S.K.
      • Sadegh-Nasseri S.
      • Benedek G.B.
      • Stern L.J.
      Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1.
      ). In DR1, P1 shows a preference for binding of aromatic side chains and aromatic dipeptides can modulate peptide binding (
      • Gupta S.
      • Höpner S.
      • Rupp B.
      • Günther S.
      • Dickhaut K.
      • Agarwal N.
      • Cardoso M.C.
      • Kühne R.
      • Wiesmüller K.-H.
      • Jung G.
      • Falk K.
      • Rötzschke O.
      Anchor side chains of short peptide fragments trigger ligand-exchange of class II MHC molecules.
      ). Simulation of a mutation in P1 revealed a long range control of conformation of the complete MHCII binding cleft (
      • Yaneva R.
      • Springer S.
      • Zacharias M.
      Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states. A molecular dynamics simulation study.
      ). Partial filling of the P1 pocket by a mutation of β Gly-86 to Tyr stabilizes the compact form of the empty DR1 dimer (
      • Sato A.K.
      • Zarutskie J.A.
      • Rushe M.M.
      • Lomakin A.
      • Natarajan S.K.
      • Sadegh-Nasseri S.
      • Benedek G.B.
      • Stern L.J.
      Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1.
      ,
      • Natarajan S.K.
      • Stern L.J.
      • Sadegh-Nasseri S.
      Sodium dodecyl sulphate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1.
      ). Mutation of Ii Met-91 to Gly, the anchor residue for binding to P1, abolishes co-isolation of Ii with a single DRα (
      • Neumann J.
      • Koch N.
      Assembly of major histocompatibility complex class II subunits with invariant chain.
      ). In the experiment presented here the Ii M91G mutant yielded impaired trimming of the DRα glycan and reduced surface exposure of DRα co-expressed with DPB1*0401 and Ii. DPβ residues that are involved in formation of P1 form a polymorphic cluster. It appears that mutations of DPβ residues of the P1 pocket lead to a conformational change that results in altered carbohydrate trimming of the DRα glycoprotein. Binding of Ii to DRαDPβ Lys-69/GGPM-(84–87) allotypes results in a structure that supports trimming of the DRα carbohydrate. We show here that the P1 pocket plays an important role for interaction of α, β, and Ii residues in the isotype-mixed MHCII heterodimer.
      Some DPβ allotypes with Glu-69 are involved in development of chronic beryllium disease, an inflammatory T cell-mediated disease. DPβ molecules with Lys-69 cannot bind beryllium and, therefore, elicit no T cell response. The polymorphism in position 69 of DPβ was shown to impact on antibody epitopes, suggesting a conformational change of the DP molecules (
      • Arroyo J.
      • Alvarez A.M.
      • Nombela C.
      • Sánchez-Pérez M.
      The role of HLA-DPβ residue 69 in the definition of antibody-binding epitope.
      ). In addition, T cell recognition is influenced by residues 69 and 84–87 of DPβ (
      • Díaz G.
      • Amicosante M.
      • Jaraquemada D.
      • Butler R.H.
      • Guillén M.V.
      • Sánchez M.
      • Nombela C.
      • Arroyo J.
      Functional analysis of HLA-DP polymorphism. A crucial role of DPβ residues 9, 11, 35, 55, 56, 69 and 84–87 in T cell allorecognition and peptide binding.
      ). The locations of the anchor positions P1 and P4 are conserved, and the polymorphic residues 69 and 84–87 determine allele-specific preferences for anchor positions. The published data together with our data suggest that the residues in position 69 and 84–87 of DPβ may have a general function in assembly of class II heterodimers.
      Expression of isotype-mixed class II peptide receptors could broaden the repertoire of a wider array of antigens presented by an individual antigen presenting cell. The structural constraints of DRαDPβ assembly presented in this paper could in addition point to an evolutionary process in generating allelic diversity within a subfamily of class II peptide receptors. Recently, it was discovered that some HLA class I alleles in the European population were from ancient origin and presumably transmitted by resident Neanderthals to immigrating humans (
      • Abi-Rached L.
      • Jobin M.J.
      • Kulkarni S.
      • McWhinnie A.
      • Dalva K.
      • Gragert L.
      • Babrzadeh F.
      • Gharizadeh B.
      • Luo M.
      • Plummer F.A.
      • Kimani J.
      • Carrington M.
      • Middleton D.
      • Rajalingam R.
      • Beksac M.
      • Marsh S.G.
      • Maiers M.
      • Guethlein L.A.
      • Tavoularis S.
      • Little A.M.
      • Green R.E.
      • Norman P.J.
      • Parham P.
      The shaping of modern immune systems by multiregional admixture with archaic humans.
      ). It is assumed that the resident Neanderthals had immune systems adapted to local pathogens in Europe. We found that the Lys-69/GGPM-(84–87) motif, which is essential for formation of DPβ to a heterodimer with DRα, is present in both Neanderthals and modern humans. The DPB1*0401 allele, which contains the Lys/GGPM motif, shows a phenotypic frequency of only 11% in sub-Saharan Africa, whereas 68% of the European population carry this allele (
      • Sidney J.
      • Steen A.
      • Moore C.
      • Ngo S.
      • Chung J.
      • Peters B.
      • Sette A.
      Five HLA-DP molecules frequently expressed in the worldwide human population share a common HLA supertypic binding specificity.
      ). The compatibility of the Neanderthal DPβ sequence in DPβDRα heterodimers suggests that a Neanderthal gene was introduced into the H. sapiens genome by admixture of the two human species. This HLA class II peptide receptor may have evolved further in ancient H. sapiens to a novel DPβ family in modern humans. If this interpretation is substantiated it may indicate that the introgression of the Neanderthal DPB allele provided a selective advantage. The nature of any such advantage is difficult to determine, but as we have shown, it may relate to the formation of novel DRADPB-mixed isotype molecules.

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