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Papers In Press, published online ahead of print January 22, 2002
Biomedical Engineering (ND20), Lerner Research Institute,Cleveland Clinic Foundation, Cleveland, OH 44195
Corresponding Author: aptes{at}bme.ri.ccf.org
Punctin (ADAMTSL-1) is a secreted molecule resembling members of the ADAMTS family of proteases. Punctin lacks the pro-metalloprotease and the disintegrin-like domain typical of this family, but contains other ADAMTS domains in precise order, including four thrombospondin type 1 repeats. Punctin is the product of a distinct gene on human chromosome 9p21-22 and mouse chromosome 4 that is expressed in adult skeletal muscle. His-tagged punctin expressed in stably-transfected High-Five insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The N-terminus is not blocked and has the sequence EEDRD…, as determined by Edman degradation, demonstrating signal peptidase processing. Recombinant epitope-tagged punctin has a calculated mass of 59991 Da, but exhibits major molecular species of 61970±6 Da and 62131±5 Da as measured by liquid chromatography electrospray mass spectrometry (LC ESMS). Punctin is a glycoprotein based on carbohydrate staining and LC ESMS glycopeptide analysis. Glycosylation occurs at a single N-linked site as demonstrated by altered electrophoretic migration of punctin expressed in the presence of tunicamycin A. Punctin contains disulfide bonds based on antibody accessibility and electrophoretic migration under reducing versus nonreducing conditions. Rotary shadowing demonstrates that punctin is hatchet-shaped, having a globular region attached to a short stem. In transfected COS-1 cells, punctin is deposited in the cell substratum in a punctate fashion and is excluded from focal contacts. Punctin is the first member of a novel family of ADAMTS-like proteins that may have important functions in the extracellular matrix.
J. Biol. Chem, 10.1074/jbc.M109665200
Submitted on October 5, 2001
Revised on January 17, 2002
Accepted on January 22, 2002
Punctin, a novel ADAMTS-like molecule (ADAMTSL-1) in extracellular matrix
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