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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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Related in: MedlinePlus

DMA and DMB mRNA levels are reduced in 2.7.93-like  mutants. Total cellular RNA from the indicated cells were analyzed in  Northern blots. Full-length probes from DMA and -B cDNAs were labeled with [32P]dCTP and used for hybridization. Reprobing of Northerns with actin (not shown) indicated equal loading of RNA to all lanes.
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Figure 8: DMA and DMB mRNA levels are reduced in 2.7.93-like mutants. Total cellular RNA from the indicated cells were analyzed in Northern blots. Full-length probes from DMA and -B cDNAs were labeled with [32P]dCTP and used for hybridization. Reprobing of Northerns with actin (not shown) indicated equal loading of RNA to all lanes.

Mentions: The transfection results, described above, suggested that DM mRNA levels might be deficient in these mutants. Therefore, DM mRNA levels in the mutants were analyzed by Northern blotting (Fig. 8). These studies confirmed that DM levels were altered in the mutants, and also indicated a phenotypic difference between 2.7.93 on the one hand, and mutants 3.4.95 and 3.6.95 on the other. In 2.7.93, both DMA and DMB levels were markedly reduced, to 10–20% of the levels of progenitor 3.1.0/DR3 (Fig. 8, and data not shown). In mutants 3.4.95 and 3.6.95, DMB levels were likewise reduced to the ∼10% level, but DMA levels were only modestly reduced. Thus, the primary defect in 2.7.93 appears to be a reduction in both DMA and DMB mRNAs whereas in 3.4.95 and 3.6.95 the principal defect appears to be a reduction in DMB mRNA. These reductions in DM mRNAs provide a reasonable basis for the reductions in DM protein and the secondary manifestations of DM deficiency in the mutants, described above. It is likely that the reduction in DMα protein in 3.4.95 and 3.6.95 results in part from accelerated turnover of DMα protein beacause of the relative deficiency of the DMβ subunit, as is common when one subunit of a dimeric protein is absent (74).


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)

DMA and DMB mRNA levels are reduced in 2.7.93-like  mutants. Total cellular RNA from the indicated cells were analyzed in  Northern blots. Full-length probes from DMA and -B cDNAs were labeled with [32P]dCTP and used for hybridization. Reprobing of Northerns with actin (not shown) indicated equal loading of RNA to all lanes.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: DMA and DMB mRNA levels are reduced in 2.7.93-like mutants. Total cellular RNA from the indicated cells were analyzed in Northern blots. Full-length probes from DMA and -B cDNAs were labeled with [32P]dCTP and used for hybridization. Reprobing of Northerns with actin (not shown) indicated equal loading of RNA to all lanes.
Mentions: The transfection results, described above, suggested that DM mRNA levels might be deficient in these mutants. Therefore, DM mRNA levels in the mutants were analyzed by Northern blotting (Fig. 8). These studies confirmed that DM levels were altered in the mutants, and also indicated a phenotypic difference between 2.7.93 on the one hand, and mutants 3.4.95 and 3.6.95 on the other. In 2.7.93, both DMA and DMB levels were markedly reduced, to 10–20% of the levels of progenitor 3.1.0/DR3 (Fig. 8, and data not shown). In mutants 3.4.95 and 3.6.95, DMB levels were likewise reduced to the ∼10% level, but DMA levels were only modestly reduced. Thus, the primary defect in 2.7.93 appears to be a reduction in both DMA and DMB mRNAs whereas in 3.4.95 and 3.6.95 the principal defect appears to be a reduction in DMB mRNA. These reductions in DM mRNAs provide a reasonable basis for the reductions in DM protein and the secondary manifestations of DM deficiency in the mutants, described above. It is likely that the reduction in DMα protein in 3.4.95 and 3.6.95 results in part from accelerated turnover of DMα protein beacause of the relative deficiency of the DMβ subunit, as is common when one subunit of a dimeric protein is absent (74).

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