title: Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4.
abstract: The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed beta-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.
authors.affiliation: Genome Research Center for Birth Defects and Genetic Diseases, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Korea.
authors.forename: Moon Suk
authors.initials: MS
authors.lastname: Kim
authors.forename: Sung-Su
authors.initials: SS
authors.lastname: Kim
authors.forename: Sang Taek
authors.initials: ST
authors.lastname: Jung
authors.forename: Jung-Young
authors.initials: JY
authors.lastname: Park
authors.forename: Han-Wook
authors.initials: HW
authors.lastname: Yoo
authors.forename: Jesang
authors.initials: J
authors.lastname: Ko
authors.forename: Katalin
authors.initials: K
authors.lastname: Csiszar
authors.forename: Sang-Yun
authors.initials: SY
authors.lastname: Choi
authors.forename: Youngho
authors.initials: Y
authors.lastname: Kim
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry*; D000629:Aminopropionitrile / Q000737:chemistry; D001596:Benzylamines / Q000737:chemistry; D003300:Copper / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D008957:Models, Genetic; D010920:Placenta / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D008249:Protein-Lysine 6-Oxidase; D011809:Quinones / Q000737:chemistry; D018121:Receptors, Cytokine / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D014466:Ultraviolet Rays
chemical_list: D001596:Benzylamines; D011809:Quinones; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; C447540:lysyl tyrosylquinone; D000629:Aminopropionitrile; D003300:Copper; C030796:benzylamine; D000594:Amino Acid Oxidoreductases; C445682:LOXL4 protein, human; D008249:Protein-Lysine 6-Oxidase; D006631:Amine Oxidase (Copper-Containing); D008239:Lysine; D003545:Cysteine