Biochemical and Biophysical Research Communications
Human ADAM33: protein maturation and localization
Section snippets
Materials and methods
Reagents. CHO-K1, COS-7, and MRC-5 cell lines were obtained from the American Type Culture Collection (Rockville, MD), HEK-293-D22 cells were obtained from Canji (San Diego, CA), and human bronchial smooth muscle cells were obtained from Clonetics Cell Discovery Systems (BioWhittaker, Walkersville, MD). Human bronchus was obtained from Tissue Transformation Technologies (Edison, NJ) with the appropriate legal consent of the donor and/or the donor’s next of kin. KB8301 was from PharMingen (BD
Catalytic activity of the metalloprotease domain
Because ADAM33 substrates are currently unknown, it was not possible to test full-length, membrane-bound protein for protease activity in cell-based cleavage assays. However, it was possible to demonstrate activity using a soluble form of the enzyme in combination with α2M, a substrate known to be cleaved by most proteases [19], [20].
A cell line was developed that expressed a soluble form of the ADAM33 catalytic domain with a carboxyl-terminus at aspartic acid 432 followed by a six-histidine
Discussion
ADAM33 contains five potential sites for asparagine-linked (N-linked) oligosaccharide modification; two sites within the pro-domain, two sites within the catalytic domain, and one site within the disintegrin domain [17 and computer analysis not shown]. Analysis of full-length ADAM33 for oligosaccharide content indicated that the immature protein consisted of predominantly high-mannose or hybrid oligosaccharides. As the protein matured, some but perhaps not all of the oligosaccharides were
Acknowledgements
The authors thank Dr. C.W. McNemar (Schering-Plough Research Institute, SPRI) for determination of N-terminal amino acids of recombinantly expressed ADAM33 and C. Rizzo (SPRI) for procurement of the human bronchus tissue. We thank Dr. L. Xiao and Dr. V. Madison (SPRI) for help in designing peptides for antibody generation and Dr. R. Ralston (Canji) for the gift of HEK-293-D22 cells. We thank Dr. C. Carlson (Covance Research Products Inc.), Dr. S.-E. Yen (Zymed Laboratories Inc.), and G. Ruiz
References (25)
- et al.
The ADAM gene family: surface proteins with adhesion and protease activity
Trends Genet.
(2000) - et al.
Tumor necrosis factor-α-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1)
J. Biol. Chem.
(2001) - et al.
Tumor necrosis factor-α-converting enzyme mediates the inducible cleavage of fractalkine
J. Biol. Chem.
(2001) - et al.
The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein
J. Biol. Chem.
(2001) - et al.
Evidence that tumor necrosis factor α converting enzyme is involved in regulated α-secretase cleavage of the Alzheimer amyloid protein precursor
J. Biol. Chem.
(1998) - et al.
Evidence for a role of a tumor necrosis factor-α (TNF-α)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival
J. Biol. Chem.
(1999) - et al.
Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch
J. Biol. Chem.
(1999) - et al.
Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity
J. Biol. Chem.
(1999) - et al.
Intracellular maturation of the mouse metalloprotease disintegrin MDC15
J. Biol. Chem.
(1998) - et al.
Identification and characterization of novel mouse and human ADAM33s with potential metalloprotease activity
Gene
(2002)