Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8.
ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.
Naus
Silvia
S
Entwicklungsbiologie und Molekulare Pathologie, W7, Universität Bielefeld, D-33615 Bielefeld, Germany.
Reipschläger
Simone
S
Wildeboer
Dirk
D
Lichtenthaler
Stefan F
SF
Mitterreiter
Stefan
S
Guan
Ziqiang
Z
Moss
Marcia L
ML
Bartsch
Jörg W
JW
D051722:ADAM Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000072197:ADAM10 Protein; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D002478:Cells, Cultured; D019483:Disintegrins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D019636:Neurodegenerative Diseases / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology
D016564:Amyloid beta-Protein Precursor; D019483:Disintegrins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D053829:Amyloid Precursor Protein Secretases; D045726:Metalloproteases; D051722:ADAM Proteins; C068490:ADAM8 protein, human; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human