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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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DM protein levels are reduced in 2.7.93-like mutants.Whole  cell lysates from the indicated cells and somatic cell hybrids were run in  denaturing SDS-PAGE and analyzed in Western immunoblots by staining  with 8338H, a rabbit polyclonal anti-DMα antiserum which recognizes  HLA-DMα monomer, as described in Materials and Methods.
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Figure 7: DM protein levels are reduced in 2.7.93-like mutants.Whole cell lysates from the indicated cells and somatic cell hybrids were run in denaturing SDS-PAGE and analyzed in Western immunoblots by staining with 8338H, a rabbit polyclonal anti-DMα antiserum which recognizes HLA-DMα monomer, as described in Materials and Methods.

Mentions: The T2 complementation results indicate that the lesion(s) in these new mutants map outside the HLA class II region and thus are neither in the HLA-DM structural genes nor in cis-acting DM regulatory elements. Because the phenotype of these mutants resembles that of DM mutants, these mutants might have regulatory lesions affecting the expression of DM which could explain their unusual phenotype. Western immunoblots of lysates from the mutants and their progenitors were stained with rabbit polyclonal antisera against DMα (Fig. 7) and DMβ (data not shown) to assess relative levels of DM protein expression. Mutant 2.7.93 expresses no detectable DMα monomer; mutants 3.6.95 and 3.4.95 exhibit significantly reduced (1/5 to 1/10 of wild type) levels of DMα monomer (Fig. 7). Western blots with rabbit polyclonal antiserum raised against DMβ cytoplasmic tail similarly reveal that mutants 2.7.93, 3.6.95, and 3.4.95 have reduced DMβ monomer (data not shown). This suggested that the phenotype of these mutants could be explained by deficient DM expression.


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)

DM protein levels are reduced in 2.7.93-like mutants.Whole  cell lysates from the indicated cells and somatic cell hybrids were run in  denaturing SDS-PAGE and analyzed in Western immunoblots by staining  with 8338H, a rabbit polyclonal anti-DMα antiserum which recognizes  HLA-DMα monomer, as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: DM protein levels are reduced in 2.7.93-like mutants.Whole cell lysates from the indicated cells and somatic cell hybrids were run in denaturing SDS-PAGE and analyzed in Western immunoblots by staining with 8338H, a rabbit polyclonal anti-DMα antiserum which recognizes HLA-DMα monomer, as described in Materials and Methods.
Mentions: The T2 complementation results indicate that the lesion(s) in these new mutants map outside the HLA class II region and thus are neither in the HLA-DM structural genes nor in cis-acting DM regulatory elements. Because the phenotype of these mutants resembles that of DM mutants, these mutants might have regulatory lesions affecting the expression of DM which could explain their unusual phenotype. Western immunoblots of lysates from the mutants and their progenitors were stained with rabbit polyclonal antisera against DMα (Fig. 7) and DMβ (data not shown) to assess relative levels of DM protein expression. Mutant 2.7.93 expresses no detectable DMα monomer; mutants 3.6.95 and 3.4.95 exhibit significantly reduced (1/5 to 1/10 of wild type) levels of DMα monomer (Fig. 7). Western blots with rabbit polyclonal antiserum raised against DMβ cytoplasmic tail similarly reveal that mutants 2.7.93, 3.6.95, and 3.4.95 have reduced DMβ monomer (data not shown). This suggested that the phenotype of these mutants could be explained by deficient DM expression.

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