Novartis Foundation Symposium Edited by GregoIy R. Bock, Joan M a s h Copyright 0 1991 by Ciba Foundation
Latent forms of TGF-P: molecular structure and mechanisms of activation Kohei Miyazono and Carl-Henrik Heldin
Ludwig Institute for Cancer Research, Box 595 Biomedical Center, S-751 24 Uppsala, Sweden
Abstract: TGF-P proteins are produced as latent, high molecular weight complexes. 'The latent form of 'IGF-PI (LTGF-PI) in human platelets comprises three components: the mature TGF-01 molecule, the N-terminal remnant of the TGF-P1 precursor in dimeric form and a novel component denoted TGF-PI-binding protein (TW-PI-BP). Recombinant TGF-PI expressed in CHO cells, which lacks TGF-PI-BP, is also produced as a latent form. Thus, the N-terminal remnant of the TGI;-P1 precursor is sufficient for TGF-PI latency, and it was denoted TGF-PI latsncy-associated peptide (TGF-fll-LAP). The cDNA for TGF-PI-BP has been cloned. I t is mainly composed of two different kinds of repeat sequences, i.e. 16 epidermal growth factor-like repeats and three copies of a cysteine-rich repeat hitherto not found in other proteins. The function of TGF-PI-BP remains to be elucidated. Activation ol' L-TCiF-P can be achieved by different chemical and enzymic treatments, or by incubation with certain cell types. Understanding of the physiological activation rnechanisms and the in vivo roles of L-TGF-P will be important for future clinical applications of TGF-P.
1991 Clinical applications Symposium 157) [I 81-92
of'
TCF-fi. Wiley, Chichesler (Ciha Foundation
TGF-P activity is mediated by a family of structurally related multifunctional polypeptides, which are produced by many different cell types (reviewed in Roberts & Sporn 1990, Lyons & Moses 1990). TGF-PI and TGF-P2 have been purified from mammalian cells and tissues, whereas TGF-P3, 4 and 5 have been cloned by low stringency hybridization from mammalian, chicken and frog cDNA libraries, respectively. TGF-PI, 2 and 3 have been found to share most of their biological activities (Graycar et a1 1989). The TGF-P family is more distantly related to several other proteins, including activins/inhibins, Miillerian inhibitory substance and bone morphogenic proteins. TGF-P proteins regulate growth and differentiation of a wide variety of cell types (Roberts & Sporn 1990). TGF-PI stimulates the growth of mesenchymal cells under certain conditions, but it acts as a potent growth inhibitor for most 81
82
Miyazono & Heldin
cell types, including epithelial cells, endothelial cells, lymphocytes and haemopoictic progenitor cells. TGF-P 1 also modulates the differentiation of different types of cells, and stimulates the overall production of extracellular matrix (Roberts & Sporn 1990). 'IGF-Pl is synthesized and secreted as a high molecular weight latent complex (Pircher et al 1984, 1986), which is important for the in vivo availability of this potent growth regulatory factor. Very high concentrations of the latent form of TGF-Pl (L-TCF-PI) were found to have some TGF-P activity, but the potency is 200-fold lower than that of TCF-Pl itself (Miyazono & Heldiri 1989). This suggests that the activation of TGF-PI from the latent complex is an important regulatory step in the action of this ubiquitous molecule (Wakefield et a1 1987). I n this paper, we describe the structure of the various forms of LTGF-P and discuss the functional roles of the different components in the latent complexes. We also discuss possible in vivo nicchanisms of activation of L-TGF-P. Striicture of I,-TC;F-B complexes TGF-PI is a 25 kDa homodimeric protein composed of 112-amino acid peptides, which are derived from 390-amino acid precursor proteins by proteolytic cleavages, linked by disulphide bonds. Purification of TGF-Pl from human platelets was initially carried out using acid-ethanol extraction as the first step, which releases TGF-p in an active form. I,-TGF-PI has also been purified from human platelets using neutral, non-denaturing conditions (Miyazono et a1 1988). The large latent complex is composed of three different subunits, i.e. the mature TGI,'-Pl dimer, the N-terminal remnant of the TGF-Pl precursor, and a novel component of 125-160 kDa denoted TGF-PI-binding protein (TGF-P1-BP). A disulphide-bonded dirner of the 40 kDa N-terminal remnant 0 1 the TGF-PI precursor is linked to a single copy of the TGF-PI-BP by disulphide bridge(s), forming a 210 kDa complex. Mature TGF-P1, cleaved from the precursor by proteolysis, is non-covalently associated with the 210 kDa complex (Miyazono et al 1988, Wakefield et al 1988) (Fig. 1). Recombinant TGF-PI, expressed in CHO cells by transfection of the TGF-P1 precursor cDNA, also occurs as a latent complex, but lacks the TGF-PI-BP (Gentry el: al 1987). Thus, the N-terminal remnant of the TGF-01 precursor is sufficient for TGF-PI latency, and this part was therefore denoted TGF-P 1 latency-associated peptide (TGF-PI -LAP). The molecular mass of recombinant L-TGF-fIl is 105 kDa, whereas that of the platelet-derived latent complex is 235 kDa: the former has been denoted the small latent complex and the latter the large laterit complex. Recombinant TGF-P2 and 3 are also produced as small latent complexes, indicating that the LAP in each of these molecules is sufficient for latency (Brown et al 1990). Small latent compkxes of TGF-8 occur not only in cells transfected with TCF-Pl precursor cDNA but also in certain cultured cells. We have recently
Latent forms of TGF-p
83
A TGF-O1-LAP (40 kDa)
Mature TGF-B1 (12 5 kDa)
TGF-O1-BP (125-190 kDa)
B
FIG. 1 . Schematic illustration of (A) large a n d (B) small latent complexes of TGF-PI (modified from Miyazono et a1 1988).
identified a glioblastoma cell line that produces several different forms of L,-TGF-P, i.e. large latent complexes of TCF-P1 and 2 , and small latent complexes of TGF-P2 (A. Olofsson et al, unpublished work 1990). The active forms of TGF-8 can also be bound and inactivated by a,-macroglobulin (O’Connor-McCourt & Wakefield 1987, Huang et al 1988). TGF-P can be dissociated from these complexes by the addition of heparin (McCaffrey et al 1989). It is possible that a,-macroglobulin acts as a scavenger for released TGF-P in vivo.
Latency-associated peptides The sequences of the N-terminal remnants of the TGF-P precursors, which are important for TGF-P latency, are less conserved than those of the C-terminal mature TGF-P peptides. TGF-P-LAPS consist of approximately 300 amino acids, and all except for TGF-P4 have N-terminal signal sequences (reviewed in Roberts & Sporn 1990). The structure of the TGF-(34 precursor is considerably different from the other forms of TGF-P, and it remains to be elucidated whether TGF-P4 also occurs as a latent complex. TGF-PI-LAP has three potential N-glycosylation sites, all of which appear to be used (Brunner et a1 1988). The carbohydrate structures contain sialic acid residues; two of them also contain mannose 6-phosphate residues (Purchio et a1
84
Miyazono & Heldin
1988). This is consistent with the observation that TGF-P1-LAP can bind to the mannose 6-phosphate/insulin-like growth factor 11 receptor (Kovacina et al 1989). It is thus possible that TGF-P1-LAP is transported to the lysosomes. Carbohydrate structures in TGF-PI-LAP may also be important for TGF-PI latency (see below). Moreover, glycosylation of the TGF-P1 precursor appears to be required for the proper secretion of TGF-61 from the producer cell (Sha et al 1989). All forms of TGF-P-LAPS, except for TGF-P2-LAP, contain Arg-Gly-Asp (KGD) sequences, a recognition motif for matrix protein receptors of the integrin family (Ruoslhhti & Pierschbacher 1987), but direct binding to inlegrins has not been demonstrated. The presence of marinose 6-phosphate residues and RGD sequences suggests that L-TGF-P or the free forms of LARS can interact with specific cellular receptors distinct from the TGF-P receptors, Thc interaction betwcen TGF-PI and TGF-PI-LAP appears to be reversible. Wakefield et al (198!)) have reported that after activation TGF-PI and TGF-0 I -LAP reassochte in a time- and concentration-dependent manner under neutral, non-denaturing conditions.
The third component o f !he large latent complex, TGF-Pl-BP TGF-p1-W is a 125-160 kDa glycoprotein, which was first purified from human platclcts as a component of the large latent TGF-PI complex (Miyazono et al 1988). Amino acid sequence analysis disclosed that it contains multiple epidermal growth factor (EGF)-1iE.e repeats (Kanzaki et al 1990). Moreover, P-hydroxylatcd asparagine residues were identified in t w o of the EGF-like repeat sequences; this post-translational modification has been found in other proteins with EGF-like repeats, and has in these cases been implicated in
Latent forms of TGF-P: molecular structure and mechanisms of activation Kohei Miyazono and Carl-Henrik Heldin
Ludwig Institute for Cancer Research, Box 595 Biomedical Center, S-751 24 Uppsala, Sweden
Abstract: TGF-P proteins are produced as latent, high molecular weight complexes. 'The latent form of 'IGF-PI (LTGF-PI) in human platelets comprises three components: the mature TGF-01 molecule, the N-terminal remnant of the TGF-P1 precursor in dimeric form and a novel component denoted TGF-PI-binding protein (TW-PI-BP). Recombinant TGF-PI expressed in CHO cells, which lacks TGF-PI-BP, is also produced as a latent form. Thus, the N-terminal remnant of the TGI;-P1 precursor is sufficient for TGF-PI latency, and it was denoted TGF-PI latsncy-associated peptide (TGF-fll-LAP). The cDNA for TGF-PI-BP has been cloned. I t is mainly composed of two different kinds of repeat sequences, i.e. 16 epidermal growth factor-like repeats and three copies of a cysteine-rich repeat hitherto not found in other proteins. The function of TGF-PI-BP remains to be elucidated. Activation ol' L-TCiF-P can be achieved by different chemical and enzymic treatments, or by incubation with certain cell types. Understanding of the physiological activation rnechanisms and the in vivo roles of L-TGF-P will be important for future clinical applications of TGF-P.
1991 Clinical applications Symposium 157) [I 81-92
of'
TCF-fi. Wiley, Chichesler (Ciha Foundation
TGF-P activity is mediated by a family of structurally related multifunctional polypeptides, which are produced by many different cell types (reviewed in Roberts & Sporn 1990, Lyons & Moses 1990). TGF-PI and TGF-P2 have been purified from mammalian cells and tissues, whereas TGF-P3, 4 and 5 have been cloned by low stringency hybridization from mammalian, chicken and frog cDNA libraries, respectively. TGF-PI, 2 and 3 have been found to share most of their biological activities (Graycar et a1 1989). The TGF-P family is more distantly related to several other proteins, including activins/inhibins, Miillerian inhibitory substance and bone morphogenic proteins. TGF-P proteins regulate growth and differentiation of a wide variety of cell types (Roberts & Sporn 1990). TGF-PI stimulates the growth of mesenchymal cells under certain conditions, but it acts as a potent growth inhibitor for most 81
82
Miyazono & Heldin
cell types, including epithelial cells, endothelial cells, lymphocytes and haemopoictic progenitor cells. TGF-P 1 also modulates the differentiation of different types of cells, and stimulates the overall production of extracellular matrix (Roberts & Sporn 1990). 'IGF-Pl is synthesized and secreted as a high molecular weight latent complex (Pircher et al 1984, 1986), which is important for the in vivo availability of this potent growth regulatory factor. Very high concentrations of the latent form of TGF-Pl (L-TCF-PI) were found to have some TGF-P activity, but the potency is 200-fold lower than that of TCF-Pl itself (Miyazono & Heldiri 1989). This suggests that the activation of TGF-PI from the latent complex is an important regulatory step in the action of this ubiquitous molecule (Wakefield et a1 1987). I n this paper, we describe the structure of the various forms of LTGF-P and discuss the functional roles of the different components in the latent complexes. We also discuss possible in vivo nicchanisms of activation of L-TGF-P. Striicture of I,-TC;F-B complexes TGF-PI is a 25 kDa homodimeric protein composed of 112-amino acid peptides, which are derived from 390-amino acid precursor proteins by proteolytic cleavages, linked by disulphide bonds. Purification of TGF-Pl from human platelets was initially carried out using acid-ethanol extraction as the first step, which releases TGF-p in an active form. I,-TGF-PI has also been purified from human platelets using neutral, non-denaturing conditions (Miyazono et a1 1988). The large latent complex is composed of three different subunits, i.e. the mature TGI,'-Pl dimer, the N-terminal remnant of the TGF-Pl precursor, and a novel component of 125-160 kDa denoted TGF-PI-binding protein (TGF-P1-BP). A disulphide-bonded dirner of the 40 kDa N-terminal remnant 0 1 the TGF-PI precursor is linked to a single copy of the TGF-PI-BP by disulphide bridge(s), forming a 210 kDa complex. Mature TGF-P1, cleaved from the precursor by proteolysis, is non-covalently associated with the 210 kDa complex (Miyazono et al 1988, Wakefield et al 1988) (Fig. 1). Recombinant TGF-PI, expressed in CHO cells by transfection of the TGF-P1 precursor cDNA, also occurs as a latent complex, but lacks the TGF-PI-BP (Gentry el: al 1987). Thus, the N-terminal remnant of the TGF-01 precursor is sufficient for TGF-PI latency, and this part was therefore denoted TGF-P 1 latency-associated peptide (TGF-PI -LAP). The molecular mass of recombinant L-TGF-fIl is 105 kDa, whereas that of the platelet-derived latent complex is 235 kDa: the former has been denoted the small latent complex and the latter the large laterit complex. Recombinant TGF-P2 and 3 are also produced as small latent complexes, indicating that the LAP in each of these molecules is sufficient for latency (Brown et al 1990). Small latent compkxes of TGF-8 occur not only in cells transfected with TCF-Pl precursor cDNA but also in certain cultured cells. We have recently
Latent forms of TGF-p
83
A TGF-O1-LAP (40 kDa)
Mature TGF-B1 (12 5 kDa)
TGF-O1-BP (125-190 kDa)
B
FIG. 1 . Schematic illustration of (A) large a n d (B) small latent complexes of TGF-PI (modified from Miyazono et a1 1988).
identified a glioblastoma cell line that produces several different forms of L,-TGF-P, i.e. large latent complexes of TCF-P1 and 2 , and small latent complexes of TGF-P2 (A. Olofsson et al, unpublished work 1990). The active forms of TGF-8 can also be bound and inactivated by a,-macroglobulin (O’Connor-McCourt & Wakefield 1987, Huang et al 1988). TGF-P can be dissociated from these complexes by the addition of heparin (McCaffrey et al 1989). It is possible that a,-macroglobulin acts as a scavenger for released TGF-P in vivo.
Latency-associated peptides The sequences of the N-terminal remnants of the TGF-P precursors, which are important for TGF-P latency, are less conserved than those of the C-terminal mature TGF-P peptides. TGF-P-LAPS consist of approximately 300 amino acids, and all except for TGF-P4 have N-terminal signal sequences (reviewed in Roberts & Sporn 1990). The structure of the TGF-(34 precursor is considerably different from the other forms of TGF-P, and it remains to be elucidated whether TGF-P4 also occurs as a latent complex. TGF-PI-LAP has three potential N-glycosylation sites, all of which appear to be used (Brunner et a1 1988). The carbohydrate structures contain sialic acid residues; two of them also contain mannose 6-phosphate residues (Purchio et a1
84
Miyazono & Heldin
1988). This is consistent with the observation that TGF-P1-LAP can bind to the mannose 6-phosphate/insulin-like growth factor 11 receptor (Kovacina et al 1989). It is thus possible that TGF-P1-LAP is transported to the lysosomes. Carbohydrate structures in TGF-PI-LAP may also be important for TGF-PI latency (see below). Moreover, glycosylation of the TGF-P1 precursor appears to be required for the proper secretion of TGF-61 from the producer cell (Sha et al 1989). All forms of TGF-P-LAPS, except for TGF-P2-LAP, contain Arg-Gly-Asp (KGD) sequences, a recognition motif for matrix protein receptors of the integrin family (Ruoslhhti & Pierschbacher 1987), but direct binding to inlegrins has not been demonstrated. The presence of marinose 6-phosphate residues and RGD sequences suggests that L-TGF-P or the free forms of LARS can interact with specific cellular receptors distinct from the TGF-P receptors, Thc interaction betwcen TGF-PI and TGF-PI-LAP appears to be reversible. Wakefield et al (198!)) have reported that after activation TGF-PI and TGF-0 I -LAP reassochte in a time- and concentration-dependent manner under neutral, non-denaturing conditions.
The third component o f !he large latent complex, TGF-Pl-BP TGF-p1-W is a 125-160 kDa glycoprotein, which was first purified from human platclcts as a component of the large latent TGF-PI complex (Miyazono et al 1988). Amino acid sequence analysis disclosed that it contains multiple epidermal growth factor (EGF)-1iE.e repeats (Kanzaki et al 1990). Moreover, P-hydroxylatcd asparagine residues were identified in t w o of the EGF-like repeat sequences; this post-translational modification has been found in other proteins with EGF-like repeats, and has in these cases been implicated in
