Two Distinct Regions of Latency-associated Peptide Coordinate Stability of the Latent Transforming Growth Factor-β1 Complex

Abstract

Transforming growth factor-␤1 (TGF-␤1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-␤1 propeptide (latency-associated peptide (LAP)), and latent TGF-␤-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-␤1 complex. Transforming growth factor-␤1 (TGF-␤1) 2 is the prototypical member of a large family of structurally related proteins, with well documented roles in cellular proliferation, differentiation, apoptosis, adhesion, and extracellular matrix deposition (1, 2). Like other family members, TGF-␤1 is synthesized as a large precursor molecule consisting of an N-terminal prodomain (latency-associated peptide (LAP)) and a C-terminal mature domain. Within this precursor, LAP acts as a multifunctional peptide capable of regulating the crucial roles TGF-␤1 plays throughout development and in the maintenance of tissue homeostasis in adult life (3-5).Initially, hydrophobic residues near the N terminus of LAP bind to mature TGF-␤1 (6), and this interaction is necessary to maintain the molecule in a conformation competent for dimerization. Two precursors are then covalently linked at sites within both the mature growth factor and LAP to form the small latent complex (SLC) (7-10). The SLC can be cleaved by proprotein convertases (11, 12), but LAP remains non-covalently associated with the mature TGF-␤1 dimer (13,14). During the secretory process, LAP also interacts covalently with latent TGF-␤-binding proteins (LTBPs) to form the large latent complex (LLC), and it is in this form that TGF-␤1 is secreted from the cell (7, 15). In the absence of LTBPs, the reactive cysteine (Cys 33 ) within LAP forms an incorrectly paired disulfide bond with a free cysteine in mature TGF-␤1, ensuring the SLC is secreted in an inactive form (10).Extracellularly, LAP confers latency to TGF-␤1 by shielding the type II receptor-binding epitope on the outer convex surface of the mature dimer (16, 17). Lacking additional secreted antagonists, this is the major point of regulation of TGF-␤1 biological activity. Interestingly, of the 33 TGF-␤ ligands, only TGF-␤1, -␤2, -␤3, myostatin, and GDF-11 (growth and differentiation factor-11) bind their propeptides with high enough affinity to confer latency (16, 18 -21). For other family members (e.g. activins and bone morphogenetic proteins), affinity for receptors is greater than affinity for propeptides, and they are secreted in an "active" form (6,22).LTBPs associate with LAP via signature 8-Cys domains, which are unique to these proteins and the structurally related molecules, fibrillin-1 and fibrillin-2 (23-25). Once secreted, LTBPs target latent TGF-␤1 to fibrillin microfibrils within the extracellular matrix. TGF-␤1 signaling is dependant upon liberation of the mature ligand from the LLC, a process mediated by activators, such as thrombospondin-1 and integrins, that bind to specific residues in LAP ( 54 LSKL and 244 RGD, respectively) (4, 26 -28). By altering the co...

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“…30 μl of mixture was mixed with 8 μl of native sample buffer (Bio-Rad, 161-0738) and then directly loaded onto a Tris-Tricine precast gel (BioRad, 345-0067). After transfer, latent and active TGF-β1 were detected on the membranes using anti-TGF-β1 antibody (R&D Systems, MAB240) as previously reported (74). In the meantime, the identity of the band for the putative active TGF-β1 was verified by mass spectrometry.…”

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