gms | German Medical Science

Background: Variants in the B3GLCT gene have been found to be protective for age-related macular degeneration (AMD) in a genome-wide association study. B3GLCT is coding for beta1-3 glucosyltransferase, which catalyzes the second step of glycosylation on thrombospondin type I repeats (TSR), forming Glc-beta1-3Fuc-O. This modification has been reported to play a role in protein secretion via an endoplasmatic reticulum quality control mechanism, although the terminal glucose is thought to be essential for secretion of only a subset of TSR-containing proteins. Since nothing is known yet about a possible protective mechanism in AMD via B3GLCT, we aimed to further explore this.

Methods: We generated B3GLCT knockout (KO) hTERT RPE1 cells using CRISPR/cas9 genome editing, and investigated whether this KO causes secretion defects of TSR-containing proteins highly expressed in the RPE. For this purpose, we evaluated gene expression of TSR-containing proteins by qPCR and subsequently we tested the presence of thrombospondin I (TSP1) and connective tissue growth factor (CTGF) in cell lysates and conditioned medium/Opti-MEM by Western blot.

Results: Gene expression analysis showed that 3 of the in total 60 TSR-containing proteins are highly expressed in hTERT RPE1 cells, which are TSP1, CTGF and cysteine rich angiogenesis inducer 61 (CYR61). CTGF and CYR61 had similar RNA levels in WT and KO cells, whereas TSP1 expression was slightly increased in the KO. On protein level, CTGF secretion was similar from WT and KO cells and TSP1 was slightly more abundant in conditioned medium from KO cells, corresponding to RNA levels.

Conclusion: KO of B3GLCT does not result in secretion defects of TSP1 and CTGF from RPE cells. Our future studies will investigate whether B3GLCT-attached glucose is required for secretion of other proteins from RPE, or whether it has additional functional roles, which could potentially be linked to AMD pathogenesis.