Novel glycosylation of HLA-DR(alpha) disrupts antigen presentation without altering endosomal localization of HLA-DR.

Format:

Book

Thesis/Dissertation

Author/Creator:

Guerra, Carolyn Bergman.

Contributor:

University of Pennsylvania.

Language:

English

Subjects (All):

Immunology.

Cytology.

0379.

0982.

Local Subjects:

0379.

0982.

Physical Description:

171 pages

Contained In:

Dissertation Abstracts International 58-11B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

The HLA-DR hemizygous B-lymphoblastoid cell line, 10.24.6, has a mutation in DR$\alpha$ (Pro96$\to$Ser) that creates a novel glycosylation site (Asn94). The mutant DR molecules in 10.24.6 remain associated with peptide fragments of invariant chain, CLIP, instead of associating with antigenic peptides. In addition, the mutation impairs the interaction of DR dimers from 10.24.6 with HLA-DM. We analyzed the mechanism by which the mutation alters antigen presentation to elucidate steps in DR biosynthesis which are required for peptide loading and to determine steps in DR biosynthesis which require an interaction with DM.

We first determined which of the alterations to the DR$\alpha$ chain in 10.24.6, the amino acid substitution or the novel glycosylation, cause the observed phenotype. After inhibiting N-linked glycosylation with tunicamycin, the differences in SDS-stability of HLA-DR molecules between progenitor 8.1.6 and mutant 10.24.6 are greatly reduced. In addition, a transfectant expressing mutant DR$\alpha$ with Pro96$\to$Ala which does not undergo novel glycosylation has normal HLA-DR antigen presentation. Together these data indicate that the novel glycan is the primary factor in the disrupted antigen presentation by the mutant DR dimers in the 10.24.6 cells.

To evaluate whether the mutant DR molecules in 10.24.6 undergo altered maturation besides the impaired release of CLIP, we used metabolic labeling and subcellular fractionation. The DR molecules from 10.24.6 showed a slight delay in transit through the Golgi apparatus and into the peptide loading compartment, but no alteration in trafficking of the mutant DR molecules was detected. In addition, the characteristics of the peptide loading compartment in 10.24.6 cells were comparable to the progenitor 8.1.6 and to a DM$\beta$-null cell line, 9.5.3. Intracellular DR accumulated in a compartment with late endosomal/early lysosomal characteristics which also contained the majority of DM. These comparisons of progenitor 8.1.6, DMS-null 9.5.3, and DR-mutant 10.24.6 demonstrate that DM is not required for the formation of the peptide loading compartment and is not required for DR to access this compartment.

Notes:

Source: Dissertation Abstracts International, Volume: 58-11, Section: B, page: 5878.

Supervisor: Elizabeth D. Mellins.

Thesis (Ph.D.)--University of Pennsylvania, 1997.

Local Notes:

School code: 0175.

ISBN:

9780591659429

Access Restriction:

Restricted for use by site license.