A polymorphic residue in the amino terminal alpha 1 hemi-domain of the mouse Ld class I molecule affects its assembly and surface expression.
In comparison to Dd and most other mouse major histocompatibility complex class I molecules, the Ld molecule is poorly expressed on the cell surface, has a lower affinity for beta 2-microglobulin and is trafficked more slowly to the cell surface. Previous studies using Ld-Dd exon-shuffled constructs and the chimeric Ddm1 molecules suggested that the Ld alpha 1 domain was responsible for this phenotype. Two constructs, one containing an Ld-Dd hemi-exon-shuffled alpha 1 exon and the other containing a Dd-Ld hemi-exon-shuffled alpha 1 exon, were inserted into either Ld or Dd to replace the intact alpha 1 exon. These constructs were transfected into mouse L cells. Flow cytometric analyses of the resulting transfectants indicate that the Dd-Ld alpha 1/Ld molecules, similar to the Dd alpha 1/Dd alpha 2/Ld molecules, were expressed at a higher level on the cell surface than either the Ld-Dd alpha 1/Ld molecules or intact Ld molecules. Analyses of the molecules in lysates suggested that a higher proportion of the Dd-Ld alpha 1/Ld molecules, like the Dd alpha 1/Dd alpha 2/Ld molecules, as compared to the Ld-Dd alpha 1/Ld and intact Ld molecules were assembled as detected by alpha 2 domain-reactive monoclonal antibodies. Pulse-chase and lysate stability studies suggested that the lower steady state levels of assembled Ld-Dd alpha 1 molecules resulted from a slower assembly rate rather than instability. Collectively, these studies suggest that residues in the amino terminal half of the Ld alpha 1 domain are responsible for its inefficient assembly, probably leading to its low cell surface expression. To determine which polymorphic residues in the amino terminal alpha 1 hemi-domain might influence this phenotype, several Ld point mutants, in which a Dd amino terminal alpha 1 hemi-domain residue was substituted into the corresponding position of Ld, were analysed. These analyses suggested that, while the residue at position 9 has only a slight effect on beta 2-microglobulin association, it has a striking effect on assembly and cell surface expression.
Talken
B L
BL
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia 65212.
Peterson
K
K
Harrison
L G
LG
Lee
D R
DR
D000818:Animals; D001483:Base Sequence; D015870:Gene Expression; D006649:Histocompatibility Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D011233:Precipitin Tests; D013329:Structure-Activity Relationship; D001613:beta 2-Microglobulin / Q000276:immunology
D006649:Histocompatibility Antigens; D001613:beta 2-Microglobulin