Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B^*5101 binding self-peptides, 27 paptides bound to HLA-B^*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B^*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B^*5102 and B^*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B^*5102) and that of Gly for Trp at residue 167 (B^*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B^*5102 or HLA-B^*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.     

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

 HLA-B^*3501 and -B^*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B^*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B^*3501 and HLA-B^*5101 molecules, we generated HLA-B^*3501 mutant molecules carrying Tyr at residue 116 (B^*3501–116Y) and tested the binding of a panel of nonamer peptides to the B^*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B^*3501–116Y molecules was much higher than that to HLA-B^*3501 and HLA-B^*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B^*3501 and HLA-B^*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B^*3501 and HLA-B^*5101 may be responsible for the low peptide binding property of the latter. Received: 18 April 1997 / Revised: 18 September 1997     

The Bw4/Bw6 difference between HLA-B*0802 and HLA-B*0801 changes the peptides endogenously bound and the stimulation of alloreactive T cells

 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. Received: 29 October 1997     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Binding of 8-mer to 11-mer peptides carrying the anchor residues to slow assembling HLA class I molecules (HLA-B*5101)

 The binding of 303 8-mer to 11-mer peptides carrying the anchor residues at P2 and the C-terminus to HLA-B^*5101 molecules was examined by a stabilization assay in which peptides were incubated with RMA-S-B^*5101 cells at 26 °C for 3 h. Analysis of the binding of these peptides to HLA-B^*5101 molecules showed that Pro and Ala at P2, and Ile, Val, and Leu at the C-terminus functioned as anchor residues, while Gly at P2 and Met at the C-terminus were weak anchors. Pro was a stronger anchor residue than Ala at P2, while Ile was the strongest anchor at the C-terminus. Among 8-mer to 11-mer peptides, the 9-mer peptides showed the strongest binding to HLA-B^*5101 molecules. This is in contrast to our recent findings that 10-mer and 11-mer peptides bind to HLA-B^*3501 molecules as effectively as 9-mer peptides. Since both HLA class I molecules have the same B-pocket and the binding peptides carry the same anchor residues, it is assumed that the structure of the F-pocket may restrict the length of binding peptides. The ability of HLA-B^*5101 binding peptides to stabilize the HLA-B^*5101 molecules was markedly lower than that of HLA-B^*3501 binding peptides to stabilize the HLA-B^*3501 molecules. It is known that HLA-B^*5101 is a slow assembling molecule, while HLA-B^*3501 assembles rapidly. The results imply that the slow assembling of HLA-B^*5101 molecules results from the low affinity of peptides to HLA-B^*5101 molecules. Received: 14 August 1996 / Revised: 8 October 1996     

HLA-B alleles of the Cayapa of Ecuador: new B39 and B15 alleles

Recent data suggest that HLA-B locus alleles can evolve quickly in native South American populations. To investigate further this phenomenon of new HLA-B variants among Amerindians, we studied samples from another South American tribe, the Cayapa from Ecuador. We selected individuals for HLA-B molecular typing based upon their HLA class II typing results. Three new variants of HLA-B39 and one new variant of HLA-B15 were found in the Cayapa: HLA-B ^*3905, HLA-B^*3906, HLA-B^*3907, and HLA-B ^*1522. A total of thirteen new HLA-B alleles have now been found in the four South American tribes studied. Each of these four tribes studied, including the Cayapa, had novel alleles that were not found in any of the other tribes, suggesting that many of these new HLA-B alleles may have evolved since the Paleo-Indians originally populated South America. Each of these 13 new alleles contained predicted amino acid replacements that were located in the peptide binding site. These amino acid replacements may affect the sequence motif of the bound peptides, suggesting that these new alleles have been maintained by selection. New allelic variants have been found for all common HLA-B locus antigenic groups present in South American tribes with the exception of B48. In spite of its high frequency in South American tribes, no evidence for variants of B48 has been found in all the Amerindians studied, suggesting that B48 may have unique characteristics among the B locus alleles.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U14756 (HLA-B ^*1522), U15683 (HLA-B ^*3905), U15639 (HLA-B ^*3906), and U15640 (HLA-B ^*3907)The names listed for these sequences were officially assigned by the WHO nomenclature Committee in September 1994, B ^*3905, and November 1994, B ^*1522, B^*3906, and B ^*3907. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1994), names will be assigned to the new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report.     

Both α and β chain polymorphisms determine the specificity of the disease-associated HLA-DQ2 molecules,with β chain residues being most influential

 We compared the peptide binding specificity of three HLA-DQ molecules; HLA-DQ(α1^*0501, β1^*0201), HLA-DQ(α1^*0201, β1^*0202), and HLA-DQ(α1^*0501, β1^*0301). The first of these molecules confers susceptibility to celiac disease and insulin-dependent diabetes mellitus, while the two latter molecules, which share either the α chain or the nearly identical β chain with HLA-DQ(α1^*0501, β1^*0201), do not predispose to these disorders. The binding of peptides was detected in biochemical binding assays as inhibition of binding of radiolabeled indicator peptides to affinity-purified HLA-DQ molecules. Binding experiments with several peptides demonstrated a clear difference in peptide binding specificity between the three HLA-DQ molecules. Further, single amino acid substitution analyses indicated that the HLA-DQ molecules have different peptide binding motifs. The experimental data were corroborated by computer modelling analysis. Our data suggest that the three HLA-DQ molecules prefer large hydrophobic residues in P1 of peptides with subtle differences in side-chain preferences. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202) both prefer large hydrophobic residues in P9, whereas HLA-DQ(α1^*0501, β1^*0301) prefers much smaller residues in this position. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202), in contrast to HLA-DQ(α1^*0501, β1^*0301), prefer negatively charged residues in P4 and P7. A less prominent P6 pocket also appears to differ between the three HLA-DQ molecules. Our results indicate that polymorphic residues of both the α and the β chain determine the peptide binding specificity of HLA-DQ(α1^*0501, β1^*0201), but that the β chain polymorphisms appears to play the most important role. The information on peptide residues which are advantageous and deleterious for binding to these HLA-DQ molecules may make possible the prediction of characteristic features of peptide that bind to HLA-DQ(α1^*0501, β1^*0201) and precipitate celiac disease. Received: 2 July 1996 / Revised: 7 August 1995     

H-2-associated control of natural cytotoxicity and hybrid resistance against RBL-5

(C57B1 × DBA)F₁ hybrids were more resistant to the inoculation of 10³ Rauscher virus-induced, serially propagated RBL-5 cells than syngeneic C57Bl recipients. Resistance was linked toH-2^di in the C57Bl backcross. Spleen cells from nonimmune (C57Bl × DBA)F₁ hybrids were significantly more reactive against RBL-5 in the natural killer (NK) cytotoxicity test in vitro than were C57Bl spleen cells. In the C57Bl backcross,H-2^dH 2^b heterozygotes were more cytotoxic than theH- 2^B homozygotes. Along with RBL-5, they were also more cytotoxic to the Moloney virus-induced YAC lymphoma (of strain A origin) and to the human ALL-derived MOLT-4 line.     

Refined peptide HLA-B*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding

 HLA-B^*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B^*3501-restricted antigens is the characterization of peptide-HLA-B^*3501 interactions. In this study, peptide-HLA-B^*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B^*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B^*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B^*3501 binding cleft. Received: 29 May 1996 / Revised: 5 August 1996     

Crucial role of N-terminal residue of binding peptides in recognition of the monoclonal antibody specific for the peptide-HLA-B5, -B35 complex

 The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B^*3501 and HLA-B^*5101 molecules carrying self-peptides. Analysis of the binding of mAb 4D12 to HLA-B^*3501 and -B^*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides and that P1 of the bound peptides critically influences its binding. The 4D12 mAb bound only to HLA-B^*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis using an HLA-B^*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent. On the other hand, 4D12 bound only to HLA-B^*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1 and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B^*3501 molecules. This was confirmed by testing the binding of mAb 4D12 to HLA-B^*3501 mutant molecules at residue 171 carrying these peptides. These results together suggest that the conformation of the A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. The present study shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex. Received: 19 June 1997 / Revised: 27 August 1997     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

Mucosal uptake and whole-body retention of dietary manganese are not altered in beta2-microglobulin knockout mice

To further examine the interrelationships between manganese and iron absorption, the mucosal uptake, initial rate of loss, wholebody retention, and tissue distribution of an orally administered ⁵⁴Mn radiotracer were compared between normal and β₂-microglobulin knockout [β₂m(-/-)] mice. These mutant mice are commonly used as a model for the study of human hemochromatosis, a hereditary ironoverload disease. Initial uptake of ⁵⁴Mn by the intestinal mucosa, the liver, and the brain was not different between the two strains. The mutant mice had much higher concentrations of nonheme and total iron in the liver, but hepatic manganese, copper, magnesium, and zinc concentrations were similar between the two strains. In summary, the mucosal uptake and whole-body retention of manganese and tissue manganese concentrations were not altered in β₂m(-/-) mice; this suggests that normal homeostasis of manganese is not affected by the altered HFE protein-β₂m complex in these mice. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. The US Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

 To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1^*0501, β1^*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1^*0201, β1^*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1^*0201, β1^*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1^*0201, β1^*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1^*0501, β1^*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1^*0501, β1^*0201) and DQ(α1^*0201, β1^*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1^*0501, β1^*0201) molecule should be helpful in the identification of CD-inducing epitopes. Received: 21 March 1997 / Revised: 28 May 1997     

Large-scale production of class I bound peptides: assigning a signature to HLA-B*1501

 A peptide-based vaccine must be bound and presented by major histocompatibility complex class I molecules to elicit a CD8⁺ T-cell response. Because class I HLA molecules are highly polymorphic, it has yet to be established how well a vaccine peptide that stimulates one individual’s CD8⁺ cytotoxic T lymphocytes will be presented by a second individual’s different class I molecules. Therefore, to facilitate precise comparisons of class I peptide binding overlaps, we uniquely combined hollow-fiber bioreactors and mass spectrometry to assign precise peptide binding signatures to individual class I HLA molecules. In applying this strategy to HLA-B^*1501, we isolated milligram quantities of B^*1501-bound peptides and mapped them using mass spectrometry. Repeated analyses consistently assign the same peptide binding signature to B^*1501; the degree of peptide binding overlap between any two class I molecules can thus be determined through comparison of their peptide signatures. Received: 3 October 1996 / Revised: 20 November 1996     

Molecular analysis of HLA-B39 subtypes

Serological studies have suggested the presence of a new HLA-B39 subtype (B39.2) in the Japanese population. To identify the new HLA-B39 subtype and compare it with an other HLA-B39 subtype (B39.1), the genes encoding HLA-B39.1 (B ^* 39013) and B39.2 (B ^* 3902) have been cloned from Japanese. We have sequenced these genes and completed the sequence of HLA-B39.1 (B ^*39011 ) gene from a Caucasian that was partially sequenced. Comparison of the sequence data revealed that B ^* 3902 and B ^* 39013 differ by three nucleotide substitutions which result in a two amino acids change at residues 63 and 67, while one silent substitution at codon 312 is found between B ^* 39011 and B ^* 39013. These results suggest that B ^* 3902 has evolved from B ^* 39013 rather than B ^* 39011.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M94051 (HLA-B^*39013), M94052 (HLA-B^*39011), and M94053 (HLA-B^*3902).     

Effects of β2–microglobulin mutations on the alpha-1 helical region of H2-Ld

 Beta-2 microglobulin (β₂m)has been shown to have an effect on the structural and functional constraints that facilitate proper class I antigen presentation. To date, no evidence has pinpointed the β₂m-specific amino acids that play an integral role in affecting structure in and around the peptide binding region of class I. To delineate β₂m amino acid positions that affect the alpha-1 helical region, we generated a series of mutant β₂m proteins bearing precise amino acid substitutions. The amino acid positions chosen were based upon previous results which demonstrated that human β₂m association with H2-L^d altered the structure of the alpha-1/alpha-2 super-domain. β₂m mutant proteins were used in β₂m exchange assays with cells expressing H2-L^d. Following exchange, cells were assayed to determine whether mutant β₂m association resulted in structural alteration of class I extracellular domains. The alteration in H2-L^d structure was evidenced by an increase in the binding of an antibody (34-1-2), specific for the alpha-1 helical region of H2-L^d. Results demonstrated that amino acid substitutions in β₂m positions 33 and 53 led to a dramatic increase in the reactivity of the alpha-1 domain-specific antibody 34-1-2. Identifying β₂m amino acid positions that influence the structure of the peptide binding region may allow for a better understanding of cellular immune responses that center upon class I/β₂m expression. Received: 18 December 1997 / Revised: 19 February 1998     

The helper T-cell repertoire of mice expressing class II major histocompatibility complex β chains in the absence of α chains

 Mutant mice generated by disrupting the H2-Aa ^b major histocompatibility complex (Mhc) gene are demonstrated here to express Aβ^b chains in the absence of α chains. These mice possess a CD4⁺ helper T cell (Th) repertoire that uses predominantly the Vβ7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the α-free Aβ molecule. As an alloantigen, the Aα-free Aβ molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal α/β heterodimers. Indeed, molecular modeling suggests a β/β homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vβ usage, toward a series of foreign α/β class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal α/β heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature. Received: 30 September 1996 / Revised: 18 October 1996     

Effects of beta-amyloid on proliferation and morphology of yeast Saccharomyces cerevisiae

Summary Deposition of beta-amyloid peptide (1–42) (βAP) in the brain is an early event linked with pathogenesis of cell injury and death in Alzheimer disease. Previous studies have demonstrated that βAP induces cytotoxicity in several types of human cells. Surprisingly, the peptide was found not only to be non toxic for yeast cells, but to stimulate growth of yeast culture. The results are consistent with βAP binding to yeast cell as illustrated by binding isotherms with the apparent dissociation constant of 8×10⁻⁷ M and B_max of 4.7×10⁴ molecules/cell.     

Singlet oxygen formation and chlorophyll a triplet excited state deactivation in the cytochrome b6f complex from Bryopsis?corticulans

We have attempted to investigate the correlation between the detergent-perturbed structural integrity of the Cyt b ₆ f complex from the marine green alga Bryopsis corticulans and its photo-protective properties, for which the nonionic detergents n-octyl-β-d-glucopyranoside (β-OG) and n-dodecyl-β-d-maltoside (β-DM), respectively, were used for the preparation of Cyt b ₆ f, and the singlet oxygen (¹O₂*) production as well as the triplet excited-state chlorophyll a (³Chl a*) formation and deactivation were examined by spectroscopic means. Near-infrared luminescence of ¹O₂ * (~1,270 nm) on photo-irradiation was detected for the β-OG preparation where the complex is mainly in oligomeric state, but not for the β-DM one in which the complex exists in dimeric form. Under anaerobic condition, photo-excitation of Chl a in the β-DM preparation generated ³Chl a* with a lower quantum yield of Φ_T ~ 0.02 and a longer lifetime of ~600 μs with respect to those as in the case of β-OG preparation, Φ_T ~ 0.12 and 200–300 μs. These results prove that the enzymatically active and intact Cyt b ₆ f complex on photo-excitation tends to produce little ³Chl a* or ¹O₂ *, which implies that the pigment–protein assembly of Cyt b ₆ f complex per se is crucial for photo-protection. F. Ma and X.-B. Chen contributed equally to this work.     

Expression of HLA class I molecules assembled with structural variants of human β2-microglobulin

 Mouse and human β₂-microglobulin (β₂m), which differ by 30% in their primary sequence, give rise to disparate levels of HLA class I heavy chain expression in transfectants of the β₂m-null FO-1 human melanoma cell line, i.e., mouse β₂m directs expression of HLA class I heavy chains that is only ∼20%–30% of that observed for heavy chains assembled with human β₂m. In this report we describe our efforts to better understand the structural basis of this regulatory phenomenon. Initial insight into the importance of the N-terminus of β₂m on HLA expression came from studies with FO-1 cells transfected with chimeric (human X mouse) B2m genes. Chimeric β₂m molecules containing residues 1–69 from human β₂m and residues 70–99 from mouse β₂m (designated HM- β₂m) induced expression of HLA heavy chains to a significantly greater extent (∼80% of level observed with cognate β₂m) than the reverse chimeric construct (designated MH- β₂m) (10%–15% of level observed with cognate β₂m). These data are consistent with the view that the major determinants of HLA class I heavy chain expression map to the portion of the β₂m molecule which forms the four-stranded β-pleated sheet, comprised of S1, S2, S4, and S5, and one strand of the three-stranded sheet (S3). The mapping of class I regulatory sites to the portion of β₂m containing the four-stranded β-pleated sheet supports the interpretation that the heavy chain contact residues on β₂m play the major role in regulating major histocompatibility (MHC) class I expression. To further dissect β₂m-mediated regulation of HLA class I expression, site-directed mutants of β₂m were prepared by conversion of human β₂m to the mouse sequence at individual amino acid positions within the four-stranded and three-stranded β-pleated sheets. Human to mouse amino acid substitutions were made in each divergent residue between positions 1–66, and as controls for COOH-terminal modification, several residues between positions 75 and 94. Cytofluorometry with HLA class I-specific antibodies indicated that cell surface expression of HLA class I heavy chains was largely insensitive to each of the individual substitutions. It is concluded that a combination of divergent residues mapping to positions of heavy chain contact are responsible for the differences observed in MHC class I expression by heterologous forms of β₂m. Received: 18 March 1997 / Revision: 21 April 1997