Functional role of N-glycosylation from ADAM10 in processing, localization and activity of the enzyme

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 cleaves several plasma membrane proteins. In this work, ADAM10 was found to contain high-mannose and complex-type glycans. Individual N-glycosylation site mutants S269A, T280A, S441A, T553A were constructed, and results indicated that all sites were occupied. T280A was found to accumulate in the endoplasmic reticulum as the non-processed precursor of the enzyme. Furthermore, it exhibited only residual levels of metalloprotease activity in vivo towards the L1 cell adhesion molecule, as well as in vitro, using a ProTNF-alpha peptide as substrate. S441A showed increased ADAM10 susceptibility to proteolysis. Mutation of N267, N439 and N551 did not completely abolish enzyme activity, however, reduced levels were found. ADAM10 is sorted into secretory vesicles, the exosomes. Here, a fraction of ADAM10 from exosomes was found to contain more processed N-linked glycans than the cellular enzyme. In conclusion, N-glycosylation is crucial for ADAM10 processing and resistance to proteolysis, and results suggest that it is required for full-enzyme activity.

Introduction

ADAM (a disintegrin and metalloprotease) 10 is a type I transmembrane glycoprotein that belongs to the ADAMs protein family. It is characterized by a conserved domain structure, consisting of an N-terminal signal sequence that directs the protein to the secretory pathway followed by a prodomain, a metalloprotease and a disintegrin domain, a cystein-rich region, a transmembrane domain and an SH3-enriched cytoplasmic tail [1]. In order to be catalytically active ADAM10 prodomain has to be cleaved by proprotein convertases furin and/or PC7 in the Golgi compartments [2], [3], [4].

ADAM10 metalloprotease domain is known to mediate the proteolytic cleavage of transmembrane proteins in their juxtamembrane region resulting in the release of the extracellular domain as a soluble form (shedding). ADAM10 can cleave a variety of proteins with importance in development, cell signalling and disease, such as the cell adhesion molecule L1 [5], pro-tumor necrosis factor-alpha [6], type IV collagen [7], amyloid precursor protein (APP) [8], ephrin-A2 [9], epidermal growth factor receptor [10], notch [11], [12], pro-heparin-binding epidermal growth factor [13], fractalkine [14], CD44 [15], N-cadherin [16], betacellulin [17], low-affinity immunoglobulin E receptor CD23 [18], among others. Moreover, ADAM10 is overexpressed in tumors, such as, uterine and ovarian carcinomas [19], human haematological malignancies [20], neuroblastomas [21], prostate cancer [22], gastric carcinoma cells [23], colon cancers [24] and oral squamous cell carcinoma [25] suggesting a role in tumor progression and dissemination.

ADAMs disintegrin and cystein-rich domains were described as being directly involved in cell–cell adhesion processes through interactions with integrins and other receptors [26].

ADAM10 can be found in various cellular compartments. It has been shown to colocalize with Golgi markers and was found on the cell surface [8], [27]. More recently, it was also detected in secreted vesicles identified as exosomes [27]. Exosomes are small membrane vesicles (30–100 nm diameter) secreted by various cell types as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane. Exosomes secretion has first been reported in a variety of cells, including cancer cells, neurons, B lymphocytes, T lymphocytes, dendritic cells, mast cells and platelets [28]. Exosomes secreted from tumor cells could promote cellular invasion and migration during metastasis [29].

ADAM10 like other ADAMs is N-glycosylated. It has four potential N-glycosylation sites (N-X-S/T, X≠Pro), three located in the metalloprotease domain (N267, N278 and N439) and one in the disintegrin domain (N551). Glycosylation is one of the most important post-translational modifications in newly synthesized proteins which may influence the physicochemical and biological properties of glycoproteins. N-glycosylation has been related with protein folding and quality control of glycoproteins in the endoplasmic reticulum with consequences for processing and trafficking (reviewed in [30], [31]), targeting in the secretory pathway, namely, to the lysosome (via the mannose-6-phosphate receptor) [32], dynamics of plasma membrane proteins [33], [34], [35], cell–cell interactions, e.g., during metastases formation [36] or inflammation [36], [37], among others.

In cancer, aberrant glycosylation, particularly of cell surface glycoconjugates, is a hallmark associated with the disease [36]. Altered glycosylation is the consequence of changes in glycosyltransferase expression and localization along the secretory pathway and also depends on the availability of sugar nucleotide donors in the Golgi lumen.

In the present work, the four potential N-glycosylation sites of bADAM10 were found to be occupied. By mutating each of those sites we observed that glycans from N278 were crucial for the intracellular processing of bADAM10, with the enzyme being accumulated as the precursor in the endoplasmic reticulum. Concerning N439, the N-glycans protected against proteolytic cleavage. N-Glycans from each mutant were required for full in vivo activity. On the other hand, a fraction of human endogenous ADAM10 from exosomes was found to contain more processed N-linked glycans than the cellular counterpart.