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Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p.

Fling SP, Rak J, Muczynski KA, Arp B, Pious D - J. Exp. Med. (1997)

Bottom Line: However, we show that the defects in two of these new mutants do not map to the DM locus.These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules.The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

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Mutant 2.7.93 expresses unstable class II dimers,  and the defects in dimer stability  in mutant 2.7.93 and DM mutant 9.5.3 are complementary.  Cell lysates from mutant 2.7.93,  and the indicated control cell  lines, DMA mutant 2.2.93 and  DMB mutant 9.5.3, and lysates  from somatic cell hybrids were  run in dimer stability assays essentially as described (55). Western blots were stained with a combination of mAb DA6.147, which recognizes DRα monomer and DRα/β dimers, and HB10.A, which reacts  with DR3β1 and DR3β3 in monomeric form and in DRα/β dimers.
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Figure 3: Mutant 2.7.93 expresses unstable class II dimers, and the defects in dimer stability in mutant 2.7.93 and DM mutant 9.5.3 are complementary. Cell lysates from mutant 2.7.93, and the indicated control cell lines, DMA mutant 2.2.93 and DMB mutant 9.5.3, and lysates from somatic cell hybrids were run in dimer stability assays essentially as described (55). Western blots were stained with a combination of mAb DA6.147, which recognizes DRα monomer and DRα/β dimers, and HB10.A, which reacts with DR3β1 and DR3β3 in monomeric form and in DRα/β dimers.

Mentions: To assess the extent of cognate peptide binding to class II molecules in these new mutants, we analyzed the stability of their class II dimers in SDS. Cognate peptide binding to class II molecules induces conformational changes in class II dimers which enhance their stability in SDS (61–63). Thus, impaired cognate peptide binding in DM mutants results in the expression of SDS unstable class II dimers. Class II dimers from 2.7.93, like class II DR dimers from DM mutants, dissociate into DRα and DRβ monomers in SDS-PAGE, whereas dimers extracted from progenitor 3.1.0/DR3 remain stable (Fig. 3). Similarly, dimers from mutants 3.6.95 and 3.4.95, but not progenitor 6.3.6/DR3, dissociate into DRα and DRβ monomers in SDS-PAGE (data not shown). Notably, these changes in dimer stability are less severe than in DM mutants (see Fig. 5 a) suggesting that the factor(s) required for class II–peptide complex assembly are significantly reduced, but not entirely absent in these mutants.


Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p.

Fling SP, Rak J, Muczynski KA, Arp B, Pious D - J. Exp. Med. (1997)

Mutant 2.7.93 expresses unstable class II dimers,  and the defects in dimer stability  in mutant 2.7.93 and DM mutant 9.5.3 are complementary.  Cell lysates from mutant 2.7.93,  and the indicated control cell  lines, DMA mutant 2.2.93 and  DMB mutant 9.5.3, and lysates  from somatic cell hybrids were  run in dimer stability assays essentially as described (55). Western blots were stained with a combination of mAb DA6.147, which recognizes DRα monomer and DRα/β dimers, and HB10.A, which reacts  with DR3β1 and DR3β3 in monomeric form and in DRα/β dimers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199108&req=5

Figure 3: Mutant 2.7.93 expresses unstable class II dimers, and the defects in dimer stability in mutant 2.7.93 and DM mutant 9.5.3 are complementary. Cell lysates from mutant 2.7.93, and the indicated control cell lines, DMA mutant 2.2.93 and DMB mutant 9.5.3, and lysates from somatic cell hybrids were run in dimer stability assays essentially as described (55). Western blots were stained with a combination of mAb DA6.147, which recognizes DRα monomer and DRα/β dimers, and HB10.A, which reacts with DR3β1 and DR3β3 in monomeric form and in DRα/β dimers.
Mentions: To assess the extent of cognate peptide binding to class II molecules in these new mutants, we analyzed the stability of their class II dimers in SDS. Cognate peptide binding to class II molecules induces conformational changes in class II dimers which enhance their stability in SDS (61–63). Thus, impaired cognate peptide binding in DM mutants results in the expression of SDS unstable class II dimers. Class II dimers from 2.7.93, like class II DR dimers from DM mutants, dissociate into DRα and DRβ monomers in SDS-PAGE, whereas dimers extracted from progenitor 3.1.0/DR3 remain stable (Fig. 3). Similarly, dimers from mutants 3.6.95 and 3.4.95, but not progenitor 6.3.6/DR3, dissociate into DRα and DRβ monomers in SDS-PAGE (data not shown). Notably, these changes in dimer stability are less severe than in DM mutants (see Fig. 5 a) suggesting that the factor(s) required for class II–peptide complex assembly are significantly reduced, but not entirely absent in these mutants.

Bottom Line: However, we show that the defects in two of these new mutants do not map to the DM locus.These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules.The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics and Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

Show MeSH
Related in: MedlinePlus