Patricia Hernández López¹, Ana B. Valverde Guirao², Gonzalo Rubio Pedraza³, Carmen Botella⁴, Salvador Ibáñez Micó⁵, Trinidad Hernández Caselles⁶

(1) Department of Biochemistry and Molecular Biology (B) and Immunology, Universidad de Murcia, IMIB-Arrixaca, 30500, España (Región de Murcia)
(2) Department of Biochemistry and Molecular Biology (B) and Immunology, Universidad de Murcia, IMIB-Arrixaca, España (Región de Murcia)
(3) INMUNOLOGÍA E INMUNOTOLERANCIA EN TRASPLANTES Y ENFERMEDADES DE BASE INMUNOLÓGICA, IMIB-Arrixaca, España
(4) Immunology Service, Virgen de la Arrixaca University Clinic Hospital, IMIB-Arrixaca, España (Región de Murcia)
(5) Pediatric Neurology Unit, Virgen de la Arrixaca University Clinic Hospital, España (Región de Murcia)
(6) INVESTIGACIÓN EN PEDIATRÍA, IMIB-Arrixaca, España

Congenital disorders of glycosylation (CDG) are a heterogeneous group of rare genetic diseases which cause abnormalities in the synthesis or binding of glycans to cellular glycoproteins and glycolipids. Consequently, numerous defects appear in many organs and systems, including the immune system. Among these diseases, PMM2-CDG is the most common disorder, caused by mutations in the PMM2 gene, which involves defects in the N-glycosylation post-traslational pathway. PMM2-CDG patients may present immune dysfunction such as deterioration in neutrophil chemotaxis and poor humoral response among other disorders. In this work we to study the HLA Class II molecules expression differences between PMM2-CDG patients and healthy individuals since they are key molecules to drive complete humoral immune responses. HLA Class II molecules present highly conserved N-glycosylation sites in their structure and those glycans seem to have a role in the HLA-II expression and antigen presentation by professional antigen presenting cells. However, the specific role of the glycosylation in this immune function is not clear at this moment.

To achieve this goal, we used EBV-transformed lymphoblastoid B cell lines (B-LCL) as the main cell model and several new anti-HLA Class II antibodies, using the flow cytometry and Western blot techniques. We studied (a) the expression of HLA-I, HLA-II and other surface antigens in B-LCL, (b) their HLA genotyping and (c) the correlation of HLA-II expression with biochemical and clinical parameters of the PMM2-CDG patients.

Our results showed that B-LCL PMM2-CDG cells expressed lower levels of surface HLA-II molecules than healthy cells, however, no differences in the expression of HLA-I were found. No significant differences in the total protein were observed when detected by Western blot. These results give support to the hypothesis that defects in the N-glycosylation pathway could produce hypoglycosylated and misfolded HLA-II molecules that may suffer degradation and/or have trouble getting the cell surface. Additionally, an association between biochemical and clinical parameters and HLA-DRα expression level was observed in PMM2-CDG patients. Finally, HLA genotyping showed expression of HLA-II haplotypes shown to have low affinity binding to CLIP peptide in most of PMM2-CDG cells.

These results confirm the applicability of the new anti-HLA-II antibodies in the study of molecular differences between PMM2-CDG and healthy cells and provide new data in the understanding of CDG disorders.