Search

Allotypes from four divergent HLA-B families (B8, B15, B16, and B27) were compared for their inhibition of cytolysis by NK cells expressing the NKB1 receptor. Allotypes differing solely at the Bw4/Bw6 region were examined as were a more divergent subset of B15 allotypes. The capacity to interact with NKB1 correlated precisely with possession of a Bw4 sequence motif at residues 77-83, whereas no correlation was made with the peptide-binding specificities of two Bw4 and four Bw6 allotypes of the B15 family. HLA-B allotypes having four different Bw4 motifs were examined and all interact with NKB1. In contrast, HLA-A allotypes, which have a Bw4 motif identical with one of those present in HLA-B, do not. Mutation at leucine 82 and arginine 83, the residues common to Bw4 motifs, shows they contribute to NKB1 interaction but are not essential. Three types of polymorphism are implicated in formation of the ligand recognized by NKB1: ones shared by Bw4 motifs; ones distinguishing Bw4 motifs; and ones outside the Bw4/Bw6 region that distinguish HLA-B from HLA-A.

Serological heterogeneity in HLA-B46 antigens has been described. Previous studies have identified B*4601 as the allele encoding the "strong" B46 antigen found in Chinese populations. Serological characterization of a Vietnamese family revealed a "weak" B46 antigen. Complementary DNA for the HLA-A, B and C alleles of three family members were cloned and the coding regions sequenced. The allele encoding the weak B46 antigen has the same coding sequence as B*4601, demonstrating that the antigenic differences are not due to polymorphism in the amino acid sequence of the HLA-B heavy chain. The recently described HLA-B*1525 and HLA-Cw*0403 alleles were also found to segregate in this Vietnamese family.

Previously, we reported overlap in the repertoires of peptides endogenously bound by a group of HLA-B allotypes related to HLA-B7. Extending such analysis to four members of the B17 family and seven members of the B15 family shows that allotypes that share sequence identity in the alpha 1 helix of the class I heavy chain possess markedly similar peptide-binding specificities. Members of the B17 family share a preference for peptides with serine, threonine, or alanine at position 2 and aromatic residues at the carboxyl terminus. Strikingly, the presence of a segment of the B17 alpha 1 helix in B*1516 and B*1517 confers the B17-like peptide-binding motif. The strong influence of natural variation in the alpha 1 helix is exemplified by the differences in peptide-binding specificity of B15 allotypes related by conversion events that replaced segments of the alpha 1 helix. In contrast, evolutionary changes that are confined to the alpha 2 domain confer less dramatic change. They do not perturb the primary anchors of the peptide-binding motif but can modulate the specificity through development and diversification of secondary anchors. Our results, in combination with those obtained previously for other HLA-B allotypes, suggest a general trend whereby polymorphism in the alpha 1 helix is the overriding influence on peptide-binding specificity of HLA-B allotypes, while amino acid substitutions in the alpha 2 domain play a more modulatory role.

Recent studies suggest that alfalfa (Medicago sativa L.) root carbohydrate concentrations are not correlated closely with shoot regrowth, and that root N reserves may influence shoot regrowth after harvest. The objectives of this greenhouse study were to (i) determine the effects of supplemental N on alfalfa regrowth and N uptake, (ii) quantify N remobilization from roots to regrowing shoots, and (iii) determine if certain root N pools, including vegetative storage proteins (VSPs), were utilized preferentially as N sources during early shoot regrowth. Four cultivars with contrasting fall dormancy reaction were defoliated and supplied with 85 mg N (kg soil) -1 as 15 N-enriched (NH 4 ) 2 SO 4 or KNO 3 . Samples were taken immediately and once weekly for 28 d. After 28 d, plants were again defoliated and shoots, crowns, and roots of one-half of the plants sampled immediately. Intact crowns and roots of remaining plants were transplanted into sand, and shoots, crowns, and roots sampled 10 d after transplanting. Taproot N from these samples was partitioned into four specific pools. Nitrate-N uptake was greater than NH 4 -N uptake at 7 and 28 d after N application. Ten days after transplanting, 25% of crown and root total N and 35% of crown and root 15 N was remobilized to regrowing shoots. Fifty-five percent of total root N was soluble in phosphate buffer. The buffer-soluble N pool was fractionated into a low molecular weight N pool (30% of total root N), a non-VSP protein N pool (20% of total root N), and a VSP-enriched protein N pool (5% of total root N). The low molecular weight N and VSP-enriched protein N pools in roots were utilized preferentially as N sources for regrowing shoots 10 d after transplanting. Accumulation of these root N pools may be important for rapid early shoot regrowth after defoliation.

The vast majority of new human HLA class I alleles are formed by conversions between existing alleles of the same locus. A notable exception to this rule is HLA-B*4601 formed by replacement of residues 66-76 of the alpha 1 helix of B*1501 by the homologous segment of Cw*0102. This inter-locus recombination, which brings together characteristic elements of HLA-B and HLA-C structure, is shown here to influence function dramatically. Naturally processed peptides bound by B*4601 are distinct from those of its parental allotypes B*1501 and Cw*0102 and dominated by three high abundance peptides. Such increased peptide selectivity by B*4601 is unique among HLA-A,B,C allotypes. For other aspects of function, presence of the small segment of HLA-C-derived sequence in an otherwise HLA-B framework converts B*4601 to an HLA-C-like molecule. Alloreactive cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and cellular glycosidases all recognize B*4601 as though it were an HLA-C allotype. These unusual properties are those of an allotype which has frequencies as high as 20% in south east Asian populations and is associated with predisposition to autoimmune diseases and nasopharyngeal carcinoma.

HLA-B67 is an uncommon antigen that has been defined by serological crossreactivity with the HLA-B7 and HLA-B16 (B38 and B39) antigens. It is found at highest frequency in certain Oriental populations and has been best defined in the Japanese. Nucleotide sequencing of cDNA encoding B67 reveals the B*6701 allele to be a subtype of B39 which differs from B*39011 by substitution at residues 67-71 of the alpha 1 helix. In the region of difference B*6701 is identical in sequence to B7, B22, B27 and related molecules that express the epitope recognized by the ME1 monoclonal antibody. That the HLA-B67 molecule binds strongly to the ME1 antibody was demonstrated by immunoprecipitation and cell surface binding assays. Identical B*6701 nucleotide sequences were obtained for the B67 alleles isolated from 2 unrelated Japanese and 1 North American caucasoid.

A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS)

Tapasin plays a critical role in promoting peptide binding by major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum. In its absence, cell surface expression of most allotypes is significantly reduced. Two exceptions are HLA-A*0201 and HLA-B*2705. In this study, the repertoire of peptides bound endogenously by these allotypes in the absence of tapasin was examined and stability of the HLA class I/peptide complexes assessed. Similar quantities of peptides were recovered from B*2705 complexes expressed in the absence and presence of tapasin and the composition of the peptide pools were not radically different. However, the stability of B*2705 molecules expressed at the surface of tapasin-deficient cells was found to be reduced which suggests there are subtle changes to the peptide repertoire. The impact of the absence of tapasin was more dramatic for A*0201. Although equivalent levels of cell surface A*0201 are expressed in the presence and absence of tapasin, very little A*0201 glycoprotein was recovered from tapasin-deficient cells suggesting the complexes readily dissociate. Consistent with reduced stability, A*0201 complexes were found to be rapidly lost from the surface of tapasin-deficient cells. Analysis of the small quantity of endogenously bound peptides recovered from A*0201 expressed in the absence of tapasin revealed a complex mixture typical of A*0201 molecules expressed in normal cells. Therefore these molecules are unable to exploit the alternative supply of TAP-independent A*0201-binding peptides present in the endoplasmic reticulum. Loading of A*0201 with peptides from both TAP-dependent and TAP-independent sources is significantly compromised without tapasin.

This chapter provides a guide to facts, such as peptide-binding specificity, alleles, serological specificity, and cells sequenced. The peptide-binding motif of an HLA allotype describes the anchoring residues preferentially bound and their position within the peptide sequence. Dominant anchors are highlighted in boldface and are those positions where one or a few closely related amino acids exclusively occupy the position. Amino acids that are enriched at a position are shown in plain text. Residues are listed in descending order of preference. Analysis of peptide binding by HLA-DR is complicated when the individual contributions of the two functional DRB genes expressed by most haplotypes are not distinguished.

The genes that encode the HLA class I and II alloantigen are closely linked to each other on the short arm of human chromosome 6. This part of the genome constitutes the human major histocompatibility complex (MHC) and is called the HLA complex 1,2 . The HLA complex encompasses some four million base pairs of DNA and is of a size comparable to the genome of the common intestinal bacterium Escherichia coli. This chapter presents the HLA class I region, it shows the class I genes and related genes. HLA-H, -J, -K and -L are complete class 1 genes that are not expressed, that is, they are pseudo genes. The MIC gene family consists of five genes, of which MICA and MICE are expressed, MICC, MICD and MICE are pseudo genes. The unlabelled genes (short vertical bars) are fragments of class I genes. HFE is a functional class I-like gene found ∼4Mb telomeric of HLA-F. In addition to the genes, some additional genes, which are interspersed amongst the class I genes and are structurally unrelated to them are also reviewed.

HLA-B*0801 is unique among HLA-B allotypes in having dominant amino acid anchors at positions 3 and 5 of the peptide-binding motif. HLA-B*0802 is a variant of HLA-B*0801 in which the Bw6 sequence motif is replaced by a Bw4 sequence motif. This change, involving substitutions at positions 77, 80, 81, 82, and 83 of the B*08 heavy chain, is probably the result of a single evolutionary event of interallelic conversion. Moreover, the difference between B*0802 and B*0801 is sufficient to stimulate a cytotoxic T-cell response. To assess further the functional impact of the Bw4 motif on a B8 background, we compared the peptide-binding specificity of the B*0801 and B*0802 allotypes by sequencing the mixture of peptides endogenously bound to B*0802 and 12 individual peptides purified from that mixture. The HLA-B*0802 allotype, while able to bind some peptides bound by B*0801, has a broader repertoire of endogenously bound peptides than B*0801: the peptides bound by B*0802 are more variable in length and exhibit greater diversity in the carboxyl-terminal amino acid which interacts with the F pocket.

Functional properties of the B*4801 allotype were investigated using HLA class I-deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl-terminus. In an in vitro cell-cell binding assay, B*4801 binds CD8 alpha homodimers weakly due to the presence of a threonine residue at position 245 in the alpha 3 domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8 alpha homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8 alpha, alloreactive T-cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti-CD8 monoclonal antibodies. Analysis of 25 B*48-expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.

Allotypes from four divergent HLA-B families (B8, B15, B16, and B27) were compared for their inhibition of cytolysis by NK cells expressing the NKB1 receptor. Allotypes differing solely at the Bw4/Bw6 region were examined as were a more divergent subset of B15 allotypes. The capacity to interact with NKB1 correlated precisely with possession of a Bw4 sequence motif at residues 77-83, whereas no correlation was made with the peptide-binding specificities of two Bw4 and four Bw6 allotypes of the B15 family. HLA-B allotypes having four different Bw4 motifs were examined and all interact with NKB1. In contrast, HLA-A allotypes, which have a Bw4 motif identical with one of those present in HLA-B, do not. Mutation at leucine 82 and arginine 83, the residues common to Bw4 motifs, shows they contribute to NKB1 interaction but are not essential. Three types of polymorphism are implicated in formation of the ligand recognized by NKB1: ones shared by Bw4 motifs; ones distinguishing Bw4 motifs; and ones outside the Bw4/Bw6 region that distinguish HLA-B from HLA-A.

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with beta 2-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 degrees C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected suggesting empty molecules recycle to acquire peptide.

The peptide-binding specificity of HLA-Cw*0304 was determined. Sequence analysis of endogenously-bound peptides isolated from Cw*0304 expressed by LCL 721.221 (221 for short) cells transfected with Cw*0304 cDNA revealed this class I allotype preferentially binds peptides possessing alanine at position 2 and leucine or methionine at the C-terminus. One peptide isolated from Cw*0304 expressed by 221 cells has sequence identity to residues 116-126 of HLA-E. Expression of HLA-E by 221 cells was confirmed by isolation of mRNA transcripts for HLA-E*0101 and detection of beta 2-microglobulin (beta 2-m)-associated HLA-E protein.

Previous studies showed the human MHC class I heavy chain HLA-B*7301 has a sequence very divergent from other class I alleles. Despite the unusual sequence, we predicted B*7301 would retain the peptide-binding function typical of other HLA-A, B and C glycoproteins, and sequence similarity to B*2705 in a region of the peptide-binding site known as the B pocket suggested B*7301 would bind peptides with Arg at position 2. To test this hypothesis, the peptide-binding specificity of B*7301 was investigated. Sequence analysis of peptides bound endogenously by B*7301 indeed found selectivity for nonamer peptides possessing Arg at position 2 and a preference for small nonpolar residues such as Pro or Ala at the C terminus was also revealed. B*7301 therefore possesses the potential to function as a conventional antigen presenting class I glycoprotein. Functional similarities between B*7301 and B*2705 are discussed in the context of the association of B*27 subtypes with susceptibility to ankylosing sponylitis and arthritic diseases.

MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA-A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells.

Background: Polymorphism among class I molecules of the major histocompatibility complex (MHC) confers allotypic specificity on the peptides that these molecules bind and present to cytotoxic T lymphocytes. Evolution of new human HLA class I alleles usually involves gene recombination events that replace a segment of one allele with the homologous region of another. In this study, the impact of these evolutionary changes has been assessed by comparison of the peptide-binding specificities of six related HLA-B allotypes. Results Endogenous peptides bound by HLA-B ∗ 5401, HLA-B ∗ 5501, HLA-B ∗ 5502, HLA-B ∗ 5601, HLA-B ∗ 6701 and HLA-B ∗ 0702 were characterized. Despite differing by 1–9 of the amino-acid residues comprising their peptide-binding sites, all these allotypes share a dominant preference for peptides that have proline at position 2. Polymorphism results in differing selection of carboxy-terminal and secondary anchor residues, but the peptide-binding specificities are sufficiently similar that there is overlap in the repertoires of peptides bound by these allotypes. Complete sequence determination of individual peptides revealed four that could be isolated from two or more allotypes. Members of the closely related HLA-B22 family — HLA-B ∗ 5401, HLA-B ∗ 5501, HLA-B ∗ 5502 and HLA-B ∗ 5601 — show only minor differences in their peptide-binding specificities. This marked similarity is reflected at the functional level, as alloreactive cytotoxic T lymphocytes generated against HLA-B ∗ 5401 and HLA-B ∗ 5501 exhibited cross-reactive recognition. Conclusion The isolation of identical endogenously bound peptides from six HLA-B allotypes demonstrates overlap in the repertoires of peptides bound in vivo by different allotypes. We speculate that the shared preference for binding peptides with proline at position 2 reflects a selective pressure to retain this specificity, which may be based upon peptide availability in vivo. Characterization of the overlap between the repertoires of peptides bound by HLA-B allotypes could simplify the development of peptide-based vaccines that are targeted to cytotoxic T cells, as single peptides would be effective for humans of different HLA types.

The nucleotide sequence of cDNA encoding the HLA-B73 antigen was determined; it is unusually divergent, differing from other HLA-B alleles by 44-77 nucleotide substitutions. Features that distinguish the B*7301 heavy chain from other HLA-B heavy chains include multiple substitutions in the alpha 3 domain and a duplication-deletion within the transmembrane region that increases the length of B*7301 compared to other HLA-B heavy chains. The duplication-deletion is shared with subsets of B alleles from the homologous gorilla (Gogo-B) and chimpanzee (Patr-B) loci. Other unusual features of B*7301 are individually shared with certain alleles of the HLA-A, HLA-C, HLA-F, Gogo-B and Patr-B loci. The B*7301 molecules has sequence elements in common with members of the B7 crossreacting group in the alpha 1 domain and is shown to possess the ME1 epitope, which is held in common with the B7, B22, B27, B42 and B67 antigens. B*7301 has a unique cysteine at position 270 of the alpha 3 domain which appears accessible but probably does not form disulphide-bonded B*7301 dimers in cell membranes. B*7301 represents a newly discovered but ancient lineage of HLA-B alleles that appears poorly represented in the modern human population.

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 THE STRUCTURE OF MHC MOLECULES . . . . . . . . . . . . . . . . . . . . . . . . . 166 THE CHARACTERIZATION OF PEPTIDES BOUND BY MHC MOLECULES , . 173 ASSEMBLY OF MHC: PEPTIDE COMPLEXES . . . . . . . . . . . . . . . . . . . . . . 182 In Vivo Binding of Peptide to MHC Class I Molecules . . . . . . . . . . . . . . . . 183 In Vivo Binding of Peptide to MHC Class II Molecules. . . . . . . . . . . . . . . . 189 SUMMARY AND PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS)