Role of N-glycosylation on ADAM10 processing, activity and sorting to exosomes

ITQB PhD Seminars

Title: Role of N-glycosylation on ADAM10 processing, activity and sorting to exosomes

Speaker: Cristina Escrevente

Laboratory: Glycobiology

Abstract
A disintegrin and metalloprotease 10 (ADAM10) is a type I transmembrane glycoprotein with four potential N-glycosylation sites (N267, N278, N439 and N551) that it is mostly found at the plasma membrane and in secreted vesicles, known as exosomes. Exosomes are small vesicles secreted by various cell types, including tumor cells, as a consequence of fusion of endosomal compartments with the plasma membrane. The major role of ADAM10 is the proteolytic cleavage of cell surface integral membrane proteins, e.g., cell adhesion molecule L1.
To investigate the role of N-glycosylation on ADAM10 processing, stability, in vivo activity and sorting to exosomes, individual N-glycosylation site mutants S269A, T280A, S441A, T553A were constructed using the QuickChange site directed mutagenesis approach. Western blot analysis of ovarian carcinoma SKOV3 cells stably transfected with wild-type and N-glycosylation mutants of bovine ADAM10 showed that all potential glycosylation sites were occupied by high-mannose-type and complex-type oligosaccharides. T280A was found to accumulate in the endoplasmic reticulum as the non-processed precursor of the enzyme while S441A showed increased susceptibility to proteolysis. The study of bADAM10 in vivo activity towards human L1, in primary embryonic fibroblast derived from ADAM10 knockout mice (ADAM10-/- MEFs), revealed that mutation of bADAM10 N-gycosylation sites did not abolish completely enzyme activity.
Both SKOV3 cells and wild-type MEFs were found to secrete exosomes containing endogenous ADAM10. Furthermore, all bADAM10 N-glycosylation mutants that were cleaved to their mature form were detected in exosomes of SKOV3 cells and ADAM10-/- MEFs, indicating that the sorting mechanism of bADAM10 to exosomes was not affected by mutating the individual N-glycosylation sites. Nevertheless, we observed that a fraction of endogenous ADAM10 from the exosomes of SKOV3 cells had more processed N-linked glycans than the cellular enzyme. An ADAM10 cleavage product was also found specifically in these secretory vesicles as the result of a metalloprotease activity
In conclusion, N-linked oligosaccharides from N278 are required for processing of the enzyme. On the other hand, N-glycosylation is not essential for enzyme activity and sorting to the exosomes of ADAM10 mature form.

Short CV

2002 – Degree in Biology, Faculdade de Ciências e Tecnologia, Universidade de Coimbra.
2003-2004 – Research fellowship at the Laboratory of Iron Metabolism, CRCHUM, Montreal University.
2004- 2007 – Research fellowship at the Laboratory of Glycobiology, ITQB.
2007 – PhD fellowship at the Laboratory of Glycobiology, ITQB.