Comments for:
Abstract
In this study, a large number of naturally processed peptides was isolated and identified from the human diabetes-susceptible class II MHC molecules HLA-DQ8 (DQA1*0301,DQB1*0302) and from murine I-Ag7 species, both of which are expressed in genetically identical APC lines. The peptides presented during the processing of autologous proteins were highly selective in showing sequence specificity, mainly consisting of 1 or more acidic residues at their C terminus. Testing for binding to the MHC molecules revealed that the position 9 (P9) acidic residues of the peptides contributed decisively to binding. For HLA-DQ8, the P1 residue, which was also an acidic amino acid, influenced binding positively. Both HLA-DQ8 and I-Ag7 selected for common peptides that bound in the same register. There was no evidence for selection of peptides having nonspecific or promiscuous binding. Thus, diabetogenic class II MHC molecules are highly selective in terms of the peptides presented by their APCs, and this is governed by the features of their P9 anchor pocket. These results are in striking contrast to those from studies examining synthetic peptide or phage display libraries, in which many peptides were shown to bind.
Authors
Anish Suri, James J. Walters, Michael L. Gross, Emil R. Unanue
Response to Unanue, et al.
Submitter: George K. Papadopoulos, Ph.D. | gpapadop@teiep.gr
Laboratory of Biochemistry and Biophysics. Faculty of Agricultural Technology. GREECE.
Published October 3, 2005
Dear Sir,
The recent work by the group of Professor Unanue [1] and the accompanying editorial by Professor Ploegh published in JCI [2] raise a number of important issues regarding the similarity of H2-Ag7 and HLA-DQ8, the two MHC class II alleles predisposing to type 1 diabetes in mouse and man, respectively. Examination of the available crystal structures for H2-Ag7 and DQ8 (3-5), as well as available evidence from biochemical, cell biological and immunological studies (6-21) indicates that there are many structural and functional features unique to H2-Ag7, without any parallel in DQ8. In the antigen-binding groove itself, the H2-Ag7 peptide-binding motif is different from that of DQ8 in most other pockets (9, 22-24), besides pocket 9. The electrostatic surface potential of the two molecules in and around the antigen-binding groove is quite different as seen from the crystal structure papers (3-5). The propensity of pocket 1 in DQ8 for acidic residues is documented by the clustering of acidic residues at the amino-terminal positions of the isolated peptides, the proportion of acidic residues at p1 emerging from the aligned peptides (ca. 40% of total, vs. 10% expected if no preferences existed), and from binding studies (22, 23). The total absence of basic residues at p1 of DQ8, also demonstrated in earlier binding studies (22, 23), is consistent with this interpretation. By contrast, the H2-Ag7 molecule has no clustering of acidic residues at the amino-terminal positions of the isolated peptides, and has only 13% of its p1 aligned residues as acidic, while ca. 10% of them are basic (18). In fact, the crystal structure of H2-Ag7 –HEL10-24 (pH 4.4) has 14Arg as its p1 anchor, a very distinctive feature (4). The only MHC II pockets documented with a preference for basic residues are the p9 pockets of the I-E alleles, and p1 of H2-Ag7 (4, 25). Also, the well-known epitope of Insulin B9-23 in NOD mouse and type 1 diabetic patients fits into the groove of DQ8 (p1 = B13Glu) in a different register than proposed to fit in the groove of H2-Ag7 (p1= B12Val), by the authors who reported the crystal structure of this molecule (3, 4). Furthermore, arginine replacement at each position of a high-affinity binding peptide, a tested method for establishing the anchor positions of a given MHC II allele (22, 25, 26), showed no binding of arginine at pockets 1, 4, 6, and 9 of DQ8 (22). True to form, there is total absence of basic amino acids in pockets 1, 4, 6 and 9 of DQ8 in all the peptides isolated (1).
In pocket 4, DQ8 can also accept aromatic amino acids (ca. 6% of peptides) (as also shown in the crystal structure of its complex with the INSB13-21 core nonamer), which are absent however from the same pocket of H2-Ag7 (18). Likewise, in pocket 6 of DQ8 acidic residues are found with a frequency of 6%, but this frequency is doubled (ca. 12%) in H2-Ag7, perhaps due to the proximity of positively charged β9His (16).
Last but not least, there are remarkably wide variations (up to 600-fold) in the IC50 or Kd values of the same peptide in binding to H2-Ag7 as reported by different laboratories, always at different pH values (Table 1). To our knowledge, such wide variations do not exist in other MHC II alleles. We believe that these arise from a pH-dependence of the motif of H2-Ag7, due to the existence of three histidines (α68, β9, and β56) and β57Ser in and near pocket 9. Thus this allele has one motif at endosomal pH (5.0) and another at extracellular pH (7.4) motif in pockets 6 and 9 (16, 19, 21). This situation leads to dissociation of most peptides bound at the endosome, once their complexes with H2-Ag7 reach the cell surface, as already documented by several laboratories (7, 12, 17, 19). We have suggested that the peptides isolated from H2-Ag7 arise from intracellular complexes, thus exist at endosomal pH, and fulfilling the corresponding motif, that favors acidic residues at p9 and to a lesser extent at p6 (16, 19).
At neutral pH (with the above three histidines uncharged) there is a lesser tendency for acidic residues at p9, but a growing tendency for aromatic and basic residues (the wide and deep pocket 9 of at pH 7.4 would allow this) (9, 16, 19, 27). Thus few peptides could fulfill both motifs, leaving most of the H2-Ag7 molecules on the cell surface empty. These findings explain a host of other properties unique to H2-Ag7, such as the instability of the molecule at the cell surface (7, 11, 12), its variable reactivity with monoclonal antibodies depending on the peptide bound (17), the extensive self-reactivity of NOD mouse T cells (8, 10, 11, 14), and the effective protection from diabetes rendered by all H2-A alleles co-expressed at ca. equal levels with H2-Ag7 in the NOD background (14, 28-30).
All the above properties are unique to the H2-Ag7 molecule and are not to be found in either DQ8 or DQ2, the other major MHC II allele conferring susceptibility to type 1 diabetes (T1D) (31). Individuals bearing DQ8 or DQ2, for example, show no extensive self-reactivity, and many HLA-DQ alleles co-expressed with DQ8 or DQ2 have no effect on T1D incidence (the two of them together increase multiplicatively the odds), while other alleles dominantly protect from the disease. There are still many puzzles regarding the molecule of H2-Ag7, but its similarity to DQ8 is not as extensive as might seem at first sight.
George Bodinas, Ph.D.
Laboratory of Biochemistry and Biophysics
Faculty of Agricultural Technology
Epirus Institute of Technology
GR47100 Arta
GREECE
Antonis K. Moustakas, Ph.D.
Department of Organic Farming
Technological Educational Institute of Ionian Islands
GR67100 Argostoli, Cephallonia
GREECE
George K. Papadopoulos, Ph.D.*
Laboratory of Biochemistry and Biophysics
Faculty of Agricultural Technology
Epirus Institute of Technology
GR47100 Arta
GREECE
gpapadop@teiep.gr
*Corresponding author
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| Table 1 | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Binding parameters for select peptides bound to H2-Ag7 and/or HLA-DQ8 | |||||||||
| IC50 or Kd µ M (pH) | |||||||||
| Sourcea,b | Peptide sequence | MHC II allele | ambient pHc | crystal structure | H2-Ag7 | HLA-DQ8 | Ref. | ||
1 4 6 7 9 | |||||||||
| HEL 14-22 | R H G L D N Y R G | H2-Ag7 | 4.4 | yes | 0.7 (6.0) | 32 | |||
| 0.3 (7.0) | 9 | ||||||||
| 9.2 (7.5)d | 4 | ||||||||
| mGAD65 209-217 | I A P V F V L L E | H2-Ag7 | 6.6 | yes | 0.08 (10.4) | 3 | |||
| 50 (4.5) | 15 | ||||||||
| MSA 565-574 | A E Q L K T V M D D | H2-Ag7 | NA | no | 0.1 (5.0) | 33 | |||
| 0.4 (6.0) | 32 | ||||||||
| 32.0 (7.5) | 4 | ||||||||
| MM 69-78 | L T A L G T I L K K | H2-Ag7 | 7.4 | no | NA | 13 | |||
| EM 69-78 | L T A L G G I L K K | H2-Ag7 | 7.4 | no | NA | 13 | |||
| SWM 110-120 | A I I H V L H S R H P | H2-Ag7 | 7.4 | no | NA | >100 (5.5) | 13,22 | ||
| MM 110-120 | I I I E V L K K R H S | H2-Ag7 | 7.4 | no | NA | 13 | |||
| INS B13-21 | E A L Y L V C G E | HLA-DQ8 | 3.5 | yes | 0.7 (6.0) | 32 | |||
| 98.3 (7.5) | 4 | ||||||||
| 0.4 (5.5) | 34 | ||||||||
| hGAD65 253-261 | I A R F K M F P E | HLA-DQ8 | 5.4 | no | 0.4 (5.5)e | 22 | |||
| a In all sequences the putative core nonamer (p1-p9) is shown. The peptides actually used are longer. See cited sources for details. | |||||||||
| b Abbreviations: EM, equine myoglobin; HEL, hen egg-white lysozyme; hGAD, human glutamic acid decarboxylase; INS, insulin (mouse/human); MM, mouse myoglobin; NA, not available; SWM, sperm whale myoglobin. | |||||||||
| c Where a crystal structure is available, the ambient pH refers to that of crystallisation. If no crystal is available, the value reported is obtained from binding studies. In case the binding studies were performed at a different pH this is noted in parenthesis, in the Kd column. | |||||||||
| d The same work reports also a Kd value of 6.0 µM for the same peptide, without stating the pH at which the experiments were performed. | |||||||||
| e The mouse GAD65 peptide (255-269, 256Y instead of F) has an optimal proliferation concentration of 7 µM, when tested with lymphocytes from immunised NOD mice. | |||||||||