Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Highly Expressed HLA-B27 Accumulate in Subcellular Vesicular Compartments and Form Oligomers That Behave Differently From HLA-B7.
Jeanty1, Cindy, Sourisce1, Adèle, Wielgosik1, Aurore, Breban2, Maxime A., Andre1, Claudine
The HLA-B27 molecule is strongly associated with spondyloarthritis (SpA). This association has been largely studied, but mechanisms of pathology remain unclear. The HLA-B27 has an enhanced propensity to misfold and form aberrant disulfide linked heavy chain dimers in the endoplasmic reticulum (ER). Animal studies have helped to go deeper understanding the mechanisms in which HLA-B27 exerts its effects. Indeed, development of a spontaneous inflammatory disease resembling human SpA in transgenic rats is specific of HLA-B27 and correlates with high levels of expression of this molecule. Our goal was to determine if and to what extent B27 homodimers are involved in this requirement of overexpression. We therefore monitored the intracellular trafficking of HLA-B27 focusing on its overexpression level.
To perform these studies, HeLa cells transfected with HLA-B*2702, -05 and -06 proteins fused at their C-ter to Renilla Luciferase (RLuc) or YFP were used. These allowed applying the Bioluminescence Resonance Energy Transfer (BRET) technique to study HLA-B27/HLA-B27 interactions in situ. Further aspects of the HLA-B27 processing (e.g. subcellular distribution, folding state, relative abundance of mono- and homo-dimers) were studied using classical techniques, including immunoblot, microscopy and flow cytometry. Cells transfected with HLA-B*0702 fusion proteins were used as control.
Monitoring the subcellular distribution of the HLA-B-YFP fusion proteins evidenced that misfolded HLA-B27 and -B7 proteins tend to accumulate in ERGIC-type (Endoplasmic Reticulum Golgi Intermediate Compartment) intracellular vesicles. This phenomenon correlated with an increased expression of the HLA-B proteins. For both, HLA-B7 and HLA-B27, BRET signals increased rapidly in HeLa cells cotransfected with HLA-B-YFP and -RLuc proteins due to heavy chain homodimerisation. Interestingly, at high levels of vesicle formation, BRET signals for HLA-B*2702 and -05 remained steady, whereas those for HLA-B*0702 and HLA-B*2706, a subtype weakly associated with SpA, strongly decreased in these high expression conditions.
Our results reveal that at a high expression levels, differences appear in the oligomerisation state between strongly SpA-associated HLA-B27 subtypes (B*2702, B*2705) and weakly or non-associated HLA-B alleles (B*2706, B*0702). The mechanisms underlying this differential behavior, possibly implying cellular responses such as UPR, autophagy or protein degradation, remain as yet unknown and are currently under investigation. These findings open new perspectives in understanding the pathogenicity of HLA-B27 proteins.
To cite this abstract, please use the following information:
Jeanty, Cindy, Sourisce, Adèle, Wielgosik, Aurore, Breban, Maxime A., Andre, Claudine; Highly Expressed HLA-B27 Accumulate in Subcellular Vesicular Compartments and Form Oligomers That Behave Differently From HLA-B7. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :1341