Identification and characterization of Lutheran blood group glycoprotein as a new substrate of membrane-type 1 matrix metalloproteinase 1 (MT1-MMP): a systemic whole cell analysis of MT1-MMP-associating proteins in A431 cells.
Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates.
Niiya
Daigo
D
Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Egawa
Nagayasu
N
Sakamoto
Takeharu
T
Kikkawa
Yamato
Y
Shinkawa
Takashi
T
Isobe
Toshiaki
T
Koshikawa
Naohiko
N
Seiki
Motoharu
M
D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008188:Lutheran Blood-Group System / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D053511:Matrix Metalloproteinase 14 / Q000302:isolation & purification / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D008188:Lutheran Blood-Group System; D008562:Membrane Glycoproteins; D053511:Matrix Metalloproteinase 14