Processing of the Fibrillin-1 Carboxyl-terminal Domain*
- Timothy M. Ritty‡,
- Thomas Broekelmann‡,
- Clarina Tisdale‡,
- Dianna M. Milewicz§ and
- Robert P. Mecham‡¶
- From the ‡Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the §Department of Internal Medicine, University of Texas-Houston Medical School, Houston, Texas 77030
Abstract
To investigate the processing and general properties of the fibrillin-1 carboxyl-terminal domain, three protein expression constructs have been developed as follows: one without the domain, one with the domain, and one with a mutation near the putative proteolytic processing site. The constructs have been expressed in two eukaryotic model systems, baculoviral and CHO-K1. Post-translational modifications that normally occur in fibrillin-1, including glycosylation, signal peptide cleavage, and carboxyl-terminal processing, occur in the three constructs in both cell systems. Amino-terminal sequencing of secreted protein revealed leader sequence processing at two sites, a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28. Processing of the carboxyl-terminal domain could be observed by migration differences in SDS-polyacrylamide gel electrophoresis and was evident in both mammalian and insect cells. Immunological identification by Western blotting confirmed the loss of the expected region. The failure of both cell systems to process the mutant construct shows that the multi-basic sequence is the site of proteolytic processing. Cleavage of the fibrillin-1 carboxyl-terminal domain occurred intracellularly in CHO-K1 cells in an early secretory pathway compartment as demonstrated by studies with secretion blocking agents. This finding, taken with the multi-basic nature of the cleavage site and observed calcium sensitivity of cleavage, suggests that the processing enzyme is a secretory pathway resident furin-like protease.
Footnotes
-
↵* This work was supported by National Institutes of Health Grants HL53325, HL29594, HL41926 (to R. P. M.), and RO1 AR43626 (to D. M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵¶ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, Box 8228, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2254; Fax: 314-362-2252; E-mail: bmecham{at}cellbio.wustl.edu.
-
↵2 M. Raghunath, E. A. Putnam, T. M. Ritty, D. Hamstra, E-S. Park, M. Tschodrich-Rotter, R. Peters, A. Rehemtulla, and D. M. Milewicz, manuscript in preparation.
- Abbreviations:
- EGF
-
epidermal growth factor
- BFA
-
brefeldin A
- BSMC
-
bovine smooth muscle cell
- ER
-
endoplasmic reticulum
- FBC
-
fetal bovine chondrocyte
- PAGE
-
polyacrylamide gel electrophoresis
- PBS
-
phosphate-buffered saline
- PNGase
-
peptide N-glycosidase F
- FBS
-
fetal bovine serum
- bp
-
base pair
- DMEM
-
Dulbecco’s modified Eagle’s medium
- CHO
-
Chinese hamster ovary
-
- Received April 22, 1998.
- Revision received December 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
