Identification of a Major Microfibril-associated Glycoprotein-1-binding Domain in Fibrillin-2

Claudio C Werneck,Barbara Crippes Trask,Thomas J Broekelmann,Timothy M Trask,Timothy M Ritty,Fernando Segade,Robert P Mecham

doi:10.1074/jbc.m402656200

Abstract

Using yeast two-hybrid, ligand blotting, and solid phase binding assays, we have shown that microfibril-associated glycoprotein-1 (MAGP-1) interacts with the 8-cysteine motif of fibrillin-2 encoded by exon 24. Binding to this sequence was demonstrated for full-length MAGP-1 as well as for the MAGP-1 matrix-binding domain encoded by exons 7 and 8. The matrix-binding domain, but not the full-length protein, also bound to regions of fibrillin-2 defined by exons 16 and 17, exon 20, and exons 23 and 24. Interestingly, no binding was detected to sequences near the N or C terminus where MAGP-1 and MAGP-2, respectively, were shown to interact with fibrillin-1. The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of fibrillin-2. Exon 24 in fibrillin lies in the region of the molecule where mutations produce the most severe phenotypes associated with Marfan syndrome (fibrillin-1) and congenital contractural arachnodactyly (fibrillin-2). It is possible that these mutations alter the ability of fibrillin to bind MAGP-1, which may contribute to the severity of the disease.

Highlights

8-cysteine motif of fibrillin-2 encoded by exon 24
The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of fibrillin-2
The MBD of MAGP-1, a cysteine-rich region encoded by exons 7 and 8, was shown in earlier studies to be the minimal sequence in MAGP-1 capable of directing its deposition onto microfibrils in the extracellular matrix (6)

Summary

EXPERIMENTAL PROCEDURES

Materials—A partial FBN2 cDNA was provided by Dr Francesco Ramirez (Hospital for Special Surgery at Weill Medical College of Cornell University, Ithaca, NY). Production of MAGP-1 and FBN2 Fragments in Mammalian Cells— The generation of CHOK1 stable transfectants expressing NLR3e/6, Aik, and full-length bovine MAGP-1 and the protein purification procedures are described elsewhere (18, 20). Positive cultures for protein expression were determined by Western blot of Ni2ϩ-nitrilotriacetic acid-agarose-purified conditioned medium. For Western blot analysis, the membranes were blocked with 5% (w/v) nonfat dry milk, 0.1% Tween 20 in phosphate-buffered saline for 1 h at 37 °C, incubated with specific primary antibodies (1:1000) for 1 h, and washed twice with blocking agent. The blots were washed and incubated with MAGP-GST antibody diluted 1:1000 in blocking agent for 1 h at 37 °C. After three washes with wash buffer, the fibrillin-coated wells were incubated for 2 h at 37 °C with serial dilutions of MAGP-1-containing medium harvested from MAGP-1-transfected CHO cells. Bioinformatics—Primary sequence input, alignment, and conceptual translation were performed with the LASERGENE package of programs (DNASTAR, Inc., Madison, WI)

RESULTS

Ligand blot

DISCUSSION