The reactants involved in the activation of latent TGF-β by cocultures of endothelial and smooth muscle cells, retinoid-treated BAE cells, and stimulated macrophages include plasmin, cation-independent mannose 6-phosphate/ insulin-like growth factor type II receptor, tissue transglutaminase, and LTBP-1 (27
). Interactions between the reactants involved in the activation of latent TGF-β have not been characterized in cell-free systems, with the exception that plasmin can cleave LAP, destabilizing noncovalent interactions between LAP and TGF-β, and that recombinant small latent complex binds to the cell surface cationindependent mannose 6-phosphate/insulin-like growth factor type II receptor via mannose 6-phosphate residues present on LAP (16
). In this article, we have identified and characterized interactions between two reactants, LTBP-1 and tissue transglutaminase, that may be involved in latent TGF-β activation by cocultures of BAE and BSM cells as well as LPS-stimulated thioglycollate-elicited macrophages (31
). These interactions include an enzyme–substrate relationship between transglutaminase and LTBP-1 and transglutaminase-dependent anchoring of large latent complex to the ECM. In addition, a second functional domain of LTBP-1 involved in latent TGF-β activation was identified.
Matrix incorporation of LTBP-1 or large latent complex (reported by others and reproduced by us) appears to occur through transglutaminase-dependent cross-linking of LTBP-1 and matrix protein(s) (60
). We observed that both large latent complex and LTBP-1 are substrates for transglutaminase, the LTBP-1 of large latent complex contains residues involved in cross-linking large latent complex, and the inhibition of transglutaminase severely attenuates the incorporation of LTBP-1 into the matrix. Transglutaminase-reactive sites of LTBP-1S appear to be located within residues 294 and 441 of the amino terminus, as an LTBP-1S mutant truncated at its amino terminus by 441 amino acids was not cross-linked to the matrix, whereas a 293–amino acid truncated LTBP-1S retained its matrix association.
Transglutaminase is more selective for glutamine residues than acyl acceptor sites (25
). A consensus sequence for glutamine residues that serve as amine acceptor sites in transglutaminase-catalyzed cross-linking has not been described. However, LTBP-1S shares some structural features present in other transglutaminase substrates. Sequence analysis of reactive glutamine residues reveals that they are exposed in loop structures, as the glutamines tend to be surrounded by positively or negatively charged amino acids (4
). In 60% of the substrates, glutamine residues are located at the amino or carboxyl terminus. From sequence alignment analysis of human LTBP-1S and LTBP-2, rat LTBP-1 and murine LTBP-3 (28
), glutamine374 and a positively flanking amino acid within the second cysteine-rich repeat of LTBP-1S is conserved among all LTBPs, suggesting that it may be a transglutaminase reactive site. Glutamine-374 is present in ΔN293 LTBP-1S, which was incorporated into ECM but is absent in ΔN441 LTBP-1S, which appeared not to be cross-linked to ECM. Identification of the transglutaminase-reactive glutamine(s) is a current subject of investigation using biochemical and molecular approaches.
The matrix protein(s) to which LTBP-1 is cross-linked has not been identified. However, LTBP-1 has been described to colocalize with fibronectin (Taipale, J., J. Saharinen, K. Hedman, and J. Keski-Oja. 1994. Mol. Biol. Cell.
5[Suppl.]:311a) and with collagen-free fibrillar structures generated by fetal rat calvarial cells (13
). LTBP-2 appears to closely associate with elastin-associated microfibrils (22
). Thus, fibronectin and proteins of elastic fibers, such as microfibril-associated glycoprotein, are candidate proteins, as they all contain residues susceptible to transglutaminase-catalyzed isopeptide bond formation (10
In addition to localizing the transglutaminase reactivity of LTBP-1S to amino-terminal residues 294–441, LTBP-1S domains participating in latent TGF-β activation were identified. Both the amino and carboxyl regions of LTBP-1S appear to be involved in protein interactions required for activation as antibodies to these two domains abrogated TGF-β generation by cocultures of BAE and BSM cells as well as by LPS-stimulated thioglycollate-elicited macrophages. Previously, we reported that protein A–purified anti450 IgG (the antibody to the amino terminus of LTBP-1S) did not affect activation of large latent complex by stimulated macrophages (44
). This discrepancy results from the fact that in the earlier work total IgG was used, whereas in the experiments reported here affinity-purified IgG was tested. Based upon the concentration of affinity-purified anti-450 IgG required to inhibit activation of large latent complex, the amount of IgG used previously would have been insufficient to observe an effect. Anti-450 IgG also inhibited matrix association of LTBP-1 and large latent complex, suggesting that the role of the amino terminus in latent TGF-β activation may be to mediate the incorporation of large latent complex into the matrix.
We have demonstrated that the carboxyl-terminal region of LTBP-1 is required for large latent complex activation, as antibodies to this region blocked TGF-β generation (44
). The carboxyl-terminal sequence of LTBP-1 does not appear to contain sites involved in covalently attaching LTBP-1 to the ECM, as addition of these antibodies to cultures did not affect the matrix content of LTBP-1. This region does contain a putative protease-sensitive site at residue 1257 located after the third cysteine-rich repeat (28
). Cleavage at this site might facilitate activation of soluble forms of large latent complex.
Matrix incorporation of large latent complex may create a concentrated pool of large latent complex. Others have proposed that segregating pools of latent TGF-β may be important in regulating the conversion of large latent complex to mature TGF-β at specific sites (23
). Alternatively, we speculate that soluble large latent complex may be resistant to plasmin activation. It has been demonstrated that activation of latent TGF-β in fibroblast CM by plasmin requires nonphysiological levels of plasmin (35
). We also find that recombinant large latent complex is not readily activated by plasmin in solution (data not shown). Therefore, cross-linking to the matrix may sequester large latent complex that is not readily activated, and subsequent release from the matrix by proteolysis (60
) could generate a modified complex susceptible to plasmin activation.
We propose that the activation of large latent complex, to release, TGF-β occurs by the sequence of reactions illustrated in Fig. . Cross-linking large latent complex to the ECM is an early step in activation of large latent complex. From our antibody inhibition studies on latent TGF-β activation, both the amino and carboxyl domains of LTBP-1 are involved in protein interactions necessary for activation. The role of the amino terminus of LTBP-1S appears to be in the cross-linking of large latent complex to the matrix, as we observed that the amino terminus contains transglutaminase reactive sites and transglutaminase activity is required for activation (31
). We propose that matrix-bound large latent complex is released by cleaving LTBP-1S at a potential tribasic protease site (arginine415) on the carboxyl side of the transglutaminase reactive residue(s). This site has been proposed by Taipale et al. to be plasmin sensitive (60
). We hypothesize that the carboxyl domain of LTBP-1S is involved in forming noncovalent interactions, perhaps with matrix, as antibodies to this domain did not interfere with the cross-linking to the matrix but did block TGF-β generation. An additional cleavage of LTBP-1S at the carboxyl terminus (residue 1257) as proposed by Taipale et al. (60
) would release a complex with an LTBP-1 molecule containing only its core EGFlike repeats plus cysteine-rich repeats 3–4 bound to small latent complex. This proteolytic processing of large latent complex may be required for subsequent activation steps, such as binding to cell surface mannose 6-phosphate/insulin-like growth factor type II receptors (16
). We speculate that cross-linking to the matrix occurs before targeting to the cell surface mannose 6-phosphate/insulin-like factor type II receptor, as the addition of excess mannose 6-phosphate does not interfere with the matrix association of LTBP-1 or large latent complex (data not shown) but abrogates TGF-β generation (16
). Once on the cell surface, fragmented latent complex is susceptible to plasmin-dependent activation by proteolytic cleavage of LAP (27
). The TGF-β released then binds to its receptor.
Figure 10 Model of plasmindependent activation of large latent complex. A diagrammatic representation of large latent complex is presented as covalently interacting with the ECM by transglutaminase (TGase)–dependent cross-linking of LTBP-1 and matrix (more ...)
This model suggests several testable questions. These include the potential significance of the putative plasmin cleavage sites in LTBP-1S, the availability of the mannose 6-phosphate residues of LAP in the complex, and the location of plasmin-sensitive sites of LAP. It is also unknown whether large latent complexes that differ in their LTBP isoforms are activated by a similar mechanism. Answers to these questions can be approached by altering specific residues in the components of large latent complex and monitoring rates of large latent complex activation.