LTBP-2 specifically interacts with the amino-terminal region of fibrillin-1 and competes with LTBP-1 for binding to this microfibrillar protein
Introduction
Transforming growth factors-β are a family of potent multifunctional cytokines which modulate a variety of cellular and physiological processes during tissue development, morphogenesis and homeostasis (Massague, 1990, Massague, 2000, Lawrence, 2001). As general characteristics, activated TGF-βs tend to inhibit cell proliferation, stimulate matrix production and exert immunosuppressive effects (Lawrence, 2001). Excessive expression of TGF-βs has been implicated in a range of diseases including pulmonary fibrosis, glomerulonephritis, liver cirrhosis, keloid formation and cancers (Blobe et al., 2000, Elliott and Blobe, 2005). The immunosuppressive properties of many tumor types may be due in part to excess production of these cytokines (Taipale et al., 1998, Massague et al., 2000). TGF-βs are secreted from cells as small latent complexes consisting of dimers of the mature cytokine linked to its pro-region or latency associated peptide, or as large latent complexes in which the small complex is covalently linked via the latency associated peptide to a latent TGF-β binding protein (LTBP) (Rifkin, 2005, Carta et al., 2006). The LTBP appears to target the latent complex to specific sites for storage within the extracellular matrix where it awaits activation by agents such as thrombospondin, integrin αVβ6, reactive oxygen species or low pH during processes such as tissue remodeling, inflammation and wound repair (Annes et al., 2003, Annes et al., 2004).
Matrix structures important for latent TGF-β storage appear to be the fibrillin-containing microfibrils (Vehvilainen et al., 2003). These microfibrils are found in the matrix of most tissues often as a component of elastic fibers in association with elastin (Mecham and Davis, 1994, Cleary and Gibson, 1996, Kielty et al., 2002). The microfibrils are composed primarily of one or more members of the fibrillin family of large glycoproteins (fibrillins 1–3) (Sakai et al., 1986, Handford et al., 2000, Kielty et al., 2005). Defects in the fibrillin-1 gene have been linked to the common genetic disorder, Marfan syndrome, characterized by skeletal, cardiovascular, ocular and lung defects (Online Mendelian Inheritance in Man number 154700). Mutations in the fibrillin-2 gene result in a related disorder, congenital contractural arachnodactyly (Online Mendelian Inheritance in Man number 121050). Recent evidence indicates that some of the phenotypic characteristics of Marfan syndrome may be due to inappropriate activation of TGF-β during tissue development and growth (Neptune et al., 2003, Ng et al., 2004). In addition, a Marfan-like phenotype has been recently linked to mutations in the TGF-β receptor 2 (TGFBR2) gene (Mizuguchi et al., 2004). These observations suggest that the fibrillin-containing microfibrils are critical regulators of latent TGF-β complex storage and that reduction in the number of microfibrils in Marfan syndrome can lead to increased activation of the latent TGF-β in tissues, resulting in some aspects of the phenotype (Judge and Dietz, 2005).
Interestingly, fibrillins share similar structural features with the LTBP family which has four members (LTBP-1–4) and the two groups of proteins are considered to compose a superfamily. LTBPs, although smaller in size, share a structure with fibrillins characterized by tandem arrays of epidermal growth factor-like 6-cysteine motifs, interspersed with characteristic 8-cysteine motifs found only in these proteins (Sinha et al., 2002, Hyytiainen et al., 2004, Rifkin, 2005). No evidence has been forthcoming for direct binding of latent TGF-β to fibrillin. However, molecular interactions of LTBPs-1 and -4 with fibrillins-1 and -2 have recently been reported (Isogai et al., 2003), supporting the concept that LTBPs anchor latent TGF-β to the microfibrils.
In contrast to other LTBPs, LTBP-2 does not appear to bind covalently to latent TGF-β Gibson et al., 1995, Saharinen and Keski-Oja, 2000) However, LTBP-2 has been shown to be abundantly associated with fibrillin-containing microfibrils during development in tissues such as aorta and elastic ligaments (Gibson et al., 1995). Currently, the function of LTBP-2 is unknown although its critical importance in early embryogenesis has been recognized since Ltbp-2 null mouse embryos were found to die between days 3.5 and 6.5 of embryonic development (Shipley et al., 2000).
In this study we show that LTBP-2 binds specifically to fibrillin-1, but not to fibrillin-2 and that the interaction involves the carboxyl-terminal region of LTBP-2 and the amino-terminal region of fibrillin-1. Moreover, LTBP-2 competes with LTBP-1 for binding to fibrillin-1 in vitro suggesting that LTBP-2 may be a negative regulator of attachment of LTBP-1 to the microfibrils in some tissues. This idea is supported by our immunohistochemical studies which show distinctive but extensively overlapping distributions for LTBPs-1 and -2 with fibrillin-1 in developing human aorta.
Section snippets
LTBP-2 specifically binds to the amino-terminal region of fibrillin-1
An expression construct for full length human LTBP-2 with a carboxyl-terminal His6-tag, LTBP-2(H) (Fig. 1A), was made using vector pCEP-4 and expressed in 293-EBNA cells. The LTBP-2(H) protein was purified from the culture medium and analyzed by SDS-PAGE and immunoblotting under reducing and non-reducing conditions (Fig. 1B). LTBP-2(H) migrated as a doublet of protein bands with apparent molecular weights of 210 kDa and 219 kDa under reducing conditions and 170 kDa and 180 kDa under
Discussion
LTBP-2 is a matrix protein of unknown function. The domain structure of LTBP-2 clearly places the molecule in the LTBP family of proteins despite evidence that LTBP-2 does not form covalent complexes with latent TGF-β in contrast to LTBP-1, LTBP-3 and LTBP-4 (Gibson et al., 1995, Rifkin, 2005). Indeed, LTBP-2 lacks the necessary consensus sequence in the third 8-Cys motif for attachment to LAP (Saharinen and Keski-Oja, 2000). LTBP-2 null mice have proved to be uninformative as they die in early
Production of recombinant polypeptides
Recombinant human fibrillin fragments (see Fig. 1) were produced in 293-EBNA cells and purified as described previously (Hanssen et al., 2004). A cDNA for full-length human LTBP-2, corresponding to bases 212–6331 (Genbank™ accession number NM_000428), was obtained by RT-PCR from human fibroblast RNA using superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) using primer 5′-TCAGGGCCCAGAACAGATTG-3′ followed by RNaseH digestion and by amplification with sense primer
Acknowledgements
The financial support of the National Health and Medical Research Council of Australia and a University of Adelaide Faculty of Health Sciences Research Development Award is gratefully acknowledged.
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- 1
Current address: Department of Biochemistry, La Trobe University, Melbourne, Victoria 3086, Australia.