Detection of endogenous MR1B protein in human 293F cells by immunoaffinity enrichment and two-dimensional western blotting

Abstract

MR1B is a splice variant of the MHC class Ib molecule MR1 that lacks the 3 extracellular domain and has been reported to modulate MR1-mediated antigen presentation to mucosal-associated invariant T (MAIT) cells in transfected cells. Although MR1B transcripts are widely expressed, MR1B protein has been reported only in transfected overexpression systems; whether endogenous MR1B protein is present has remained unknown. Here we report the detection of endogenous MR1B protein. Immunoaffinity enrichment of 293F cell lysate (447 mg total protein) followed by two-dimensional electrophoresis and western blotting gave immunoreactive spots at the MR1A (~40 kDa) and MR1B (~27-30 kDa) reference coordinates established from recombinant cells, whose MR1 identity was confirmed by mass spectrometry. The spots were detected with two antisera raised against regions on opposite sides of the deleted segment and were absent with the corresponding pre-immune sera; detection with the C-terminal antiserum was obtained once. The 27-30 kDa spot lies on the basic side of the MR1A spot, the direction predicted for loss of the 3 domain and opposite to that predicted for a modeled, unmodified N-terminal fragment of similar mass. Mass spectrometry identified MR1 only in the recombinant reference spots, so the endogenous assignment rests on immunoreactivity and two-dimensional position. Endogenous MR1B protein is detected in 293F cells under non-overexpression conditions.