Functional role of N-glycosylation from ADAM10 in processing, localization and activity of the enzyme.
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.
Escrevente
Cristina
C
Instituto de Tecnologia Química e Biológica, Apartado 127, 2781-901 Oeiras, Portugal.
Morais
Vanessa A
VA
Keller
Sascha
S
Soares
Cláudio M
CM
Altevogt
Peter
P
Costa
Júlia
J
D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D019943:Amino Acid Substitution; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D039842:Neural Cell Adhesion Molecule L1 / Q000737:chemistry / Q000378:metabolism; D046188:Protein Modification, Translational / Q000502:physiology*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism
D008565:Membrane Proteins; D039842:Neural Cell Adhesion Molecule L1; D011498:Protein Precursors; D014409:Tumor Necrosis Factor-alpha; C061153:tumor necrosis factor precursor; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human