Transthyretin, a New Cryptic Protease

Transthyretin (TTR) is a plasma homotetrameric protein that acts physiologically as a transporter of thyroxine (T4) and retinol, in the latter case through binding to retinol-binding protein (RBP). A fraction of plasma TTR is carried in high density lipoproteins by binding to apolipoprotein AI (apoA-I). We further investigated the nature of the TTR-apoA-I interaction and found that TTR from different sources (recombinant and plasmatic) is able to process proteolytically apoA-I, cleaving its C terminus after Phe-225. TTR-mediated proteolysis was inhibited by serine protease inhibitors (phenylmethanesulfonyl fluoride, Pefabloc, diisopropyl fluorophosphate, chymostatin, and Nα-p-tosyl-l-phenylala-nine-chloromethyl ketone), suggesting a chymotrypsin-like activity. A fluorogenic substrate corresponding to an apoA-I fragment encompassing amino acid residues 223-228 (Abz-ESFKVS-EDDnp) was used to characterize the catalytic activity of TTR, including optimum reaction conditions (37 °C and pH 6.8) and catalytic constant (Km = 29 μm); when complexed with RBP, TTR activity was lost, whereas when complexed with T4, only a slight decrease was observed. Cell lines expressing TTR were able to degrade Abz-ESFKVS-EDDnp 2-fold more efficiently than control cells lacking TTR expression; this effect was reversed by the presence of RBP in cell culture media, therefore proving a TTR-specific proteolytic activity. TTR can act as a novel plasma cryptic protease and might have a new, potentially important role under physiological and/or pathological conditions. Transthyretin (TTR) is a plasma homotetrameric protein that acts physiologically as a transporter of thyroxine (T4) and retinol, in the latter case through binding to retinol-binding protein (RBP). A fraction of plasma TTR is carried in high density lipoproteins by binding to apolipoprotein AI (apoA-I). We further investigated the nature of the TTR-apoA-I interaction and found that TTR from different sources (recombinant and plasmatic) is able to process proteolytically apoA-I, cleaving its C terminus after Phe-225. TTR-mediated proteolysis was inhibited by serine protease inhibitors (phenylmethanesulfonyl fluoride, Pefabloc, diisopropyl fluorophosphate, chymostatin, and Nα-p-tosyl-l-phenylala-nine-chloromethyl ketone), suggesting a chymotrypsin-like activity. A fluorogenic substrate corresponding to an apoA-I fragment encompassing amino acid residues 223-228 (Abz-ESFKVS-EDDnp) was used to characterize the catalytic activity of TTR, including optimum reaction conditions (37 °C and pH 6.8) and catalytic constant (Km = 29 μm); when complexed with RBP, TTR activity was lost, whereas when complexed with T4, only a slight decrease was observed. Cell lines expressing TTR were able to degrade Abz-ESFKVS-EDDnp 2-fold more efficiently than control cells lacking TTR expression; this effect was reversed by the presence of RBP in cell culture media, therefore proving a TTR-specific proteolytic activity. TTR can act as a novel plasma cryptic protease and might have a new, potentially important role under physiological and/or pathological conditions. Transthyretin (TTR) 1The abbreviations used are: TTR, transthyretin; apoA-I, apolipoprotein AI; ATTR, transthyretin-related amyloidosis, DFP, diisopropyl fluorophosphate; HDL, high-density lipoprotein; HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PMSF, phenylmethylsulfonyl fluoride; RBP, retinol-binding protein; T4, thyroxine; TLCK, Nα-p-tosyl-l-lysine-chloromethyl ketone; TPCK, Nα-p-tosyl-l-phenylalanine-chloromethyl ketone; wt, wild-type. is a plasma protein of four identical subunits of ∼14 kDa (1Blake C.C.F. Geisow M.J. Oatley S.J. J. Mol. Biol. 1978; 121: 339-356Crossref PubMed Scopus (717) Google Scholar) that is mainly synthesized by the liver and the choroid plexus of the brain (2Dickson P.W. Howlett G.J. Schreiber G. J. Biol. Chem. 1985; 260: 8214-8219Abstract Full Text PDF PubMed Google Scholar). Under physiological conditions, TTR functions as a carrier for both thyroxine (T4) and retinol (vitamin A), in the latter case through binding to the retinol-binding protein (RBP) (3Monaco H.L. Biochim. Biophys. Acta. 2000; 1482: 65-72Crossref PubMed Scopus (149) Google Scholar). We have determined previously that apolipoprotein AI (apoA-I) is also a TTR ligand; physiologically, a fraction of plasma TTR circulates in high density lipoproteins (HDLs) through binding to apoA-I (4Sousa M.M. Berglund L. Saraiva M.J. J. Lipid Res. 2000; 41: 58-65Abstract Full Text Full Text PDF PubMed Google Scholar), its major protein component. The physiological meaning of this interaction remains to be explained, but it first suggested that it might be relevant in physiological conditions, namely in lipid and/or TTR metabolism. Previous evidence suggested that TTR and lipoprotein biology might be related. In the kidney, megalin, a member of the lipoprotein receptor family, is responsible for TTR tubular reabsorption (5Sousa M.M. Norden A.G.W. Jacobsen C. Willnow T.E. Christensen E.I. Thakker R.V. Verroust P.J. Moestrup S.K. Saraiva M.J. J. Biol. Chem. 2000; 275: 38176-38181Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Furthermore, TTR uptake by the liver (its major site of degradation) is sensitive to the receptor-associated protein, an inhibitor of uptake of all the ligands of the lipoprotein receptor family (6Sousa M.M. Saraiva M.J. J. Biol. Chem. 2001; 276: 14420-14425Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Several point mutations in TTR have been linked to the occurrence of TTR-related amyloidosis (ATTR), a disorder that is characterized by the extracellular systemic deposition of mutated or wild-type (wt) TTR as amyloid fibrils (7Saraiva M.J.M. Birken S. Costa P.P. Goodman D.S. J. Clin. Investig. 1984; 74: 104-119Crossref PubMed Google Scholar, 8Cornwell I.I.I. Sletten K. Jonhansson B. Westermark P. Biochem. Biophys. Res. Commun. 1988; 154: 648-653Crossref PubMed Scopus (137) Google Scholar), leading to organ dysfunction and death. The mechanisms by which soluble proteins self-assemble into a fibrillary structure are unknown. Proteolysis has been thought to play an important role in most types of amyloidoses; in several cases, an amyloid peptide is generated by proteolysis of the corresponding protein precursor (9Kisilevsky R. J. Struct. Biol. 2000; 130: 99-108Crossref PubMed Scopus (120) Google Scholar, 10Schormann N. Murrell J.R. Benson M.D. Amyloid. 1998; 5: 175-187Crossref PubMed Scopus (54) Google Scholar). C-terminal TTR peptides with proteolysis occurring between amino acid residues 42 and 59, in addition to intact protein, have been found in amyloid fibrils of some ATTR patients (11Gustavsson A. Jahr H. Tobiassen R. Jacobson D.R. Sletten K. Westermark P. Lab. Investig. 1995; 73: 703-708PubMed Google Scholar). This raised the hypothesis that proteolysis could trigger fibril formation. Amyloidogenic variants of apoA-I have also been reported (12Walsh M.T. Am. J. Pathol. 1999; 154: 11-14Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). ApoA-I, a 243-amino acid protein, is synthesized by the liver and plays a key role in the formation, metabolism and catabolism of HDL cholesterol esters. Numerous attempts have been made to determine the arrangement of the amphipathic helices of apoA-I in nascent HDL discs, as they appear to be a critical intermediate in reverse cholesterol transport; however, the tertiary arrangement of apoA-I molecules on HDL particles is not defined yet. In apoA-I-related amyloidosis, amyloid fibrils are characterized by deposition of N-terminal fragments of variable length in the mutated protein (12Walsh M.T. Am. J. Pathol. 1999; 154: 11-14Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Interestingly we identified an amyloidogenic variant of apoA-I, L178H, where fibrils presented both mutated apoA-I and wt TTR that co-localized in amyloid deposits as seen by immunohistochemistry and analysis of extracted fibrils (13de Sousa M.M. Vital C. Ostler D. Fernandes R. Pouget-Abadie J. Carles D. Saraiva M.J. Am. J. Pathol. 2000; 156: 1911-1917Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). This data further suggested that the interaction between apoA-I and TTR might be relevant not only physiologically but also in pathological conditions. In this report we further investigated the apoAI-TTR interaction and verified that TTR is able to process apoA-I proteolytically. Proteins and Protease Inhibitors—Recombinant TTR was produced in Escherichia coli D1210 transformed with pINTR plasmid carrying TTR cDNA (14Furuya H. Nakazato M. Saraiva M.J. Costa S.P. Sasaki H. Matsuo H. Goto I. Sakaki Y. Biochem. Biophys. Res. Commun. 1989; 163: 851-859Crossref PubMed Scopus (12) Google Scholar). The protein was isolated and purified as described previously (15Almeida M.R. Damas A.M. Lans M.C. Brower A. Saraiva M.J. Endocrine. 1997; 6: 309-315Crossref PubMed Scopus (85) Google Scholar). Briefly, after osmotic shock of bacteria, protein extracts were run on diethylaminoethyl (DEAE)-cellulose (Whatman) ion exchange chromatography, dialyzed, lyophilized, and isolated in native preparative Prosieve agarose (FMC Corp.) gel electrophoresis following the supplier's instructions. After electrophoresis, the TTR band was excised and electroeluted in an Elutrap system (Schleicher & Schuell) in 38 mm glycine and 5 mm Tris, pH 8.3, overnight at 50 V (4 °C). A final purification by high pressure liquid chromatography (HPLC) was performed on a Protein Pak 125 column (Waters) coupled to a high precision pump (P-302) and a halocrome-280 UV detector (Gilson), using as eluent 200 mm sodium phosphate buffer, pH 7, at a flow rate of 0.4 ml/min, and 400-μl fractions were collected. Protein standards used for calibration were bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and lysozyme (14 kDa). Serum TTR was purified as described previously (15Almeida M.R. Damas A.M. Lans M.C. Brower A. Saraiva M.J. Endocrine. 1997; 6: 309-315Crossref PubMed Scopus (85) Google Scholar). Briefly, plasma was dialyzed against 77 mm NaCl and 50 mm phosphate buffer, pH 7.6, and run on a DEAE-cellulose ion exchange column, after which protein fractions were dialyzed, lyophilized, and chromatographed on a blue Sepharose column (Amersham Biosciences). Plasma TTR was isolated by preparative electrophoresis, followed by electroelution and HPLC as described above for recombinant TTR. Serum RBP was isolated by affinity in a TTR column and saturated with 3.3 mg/ml all-trans retinol (Sigma) in ethanol as follows: (i) 25 μl of all-trans-retinol were incubated with 800 μl of RBP (1 mg/ml) at 37 °C in the dark for and retinol was from RBP by gel in ApoA-I, and diisopropyl were from and were from Pefabloc, chymostatin, Nα-p-tosyl-l-phenylalanine-chloromethyl Nα-p-tosyl-l-lysine-chloromethyl and were from TTR Proteolysis as and apoA-I at an were incubated in 50 mm Tris, pH at 37 °C for After were run on and by and TTR was with of the protease inhibitors mm or mm mm TPCK, TLCK, chymostatin, mm PMSF, DFP, and with for at 37 °C the addition of of N-terminal of the apoAI-TTR reaction was run on a to a (Amersham and with for 5 of apoA-I fragments were and N-terminal was performed on an protein matrix-assisted laser mass apoA-I were from a gel with were by with and with and and for by with and and with and After gel were with and on a mass in a C. C. S. A. PubMed Scopus Google Scholar). C-terminal of the apoA-I 200 TTR and of apoA-I were incubated in 50 mm Tris, pH overnight at 37 and the reaction was run on a to a and with of apoA-I fragments were and C-terminal was performed on an protein M. G. Biochem. PubMed Scopus Google Scholar). were used to activity of TTR. 25 of recombinant TTR were run After electrophoresis, were incubated in for and overnight at 37 °C in 50 mm Tris, 200 mm 5 mm and were with in and acid for and in and acid for proteins were to a (Amersham with in phosphate incubated for at with in and incubated for at with with in The was using (Sigma) as of proteolytic activity was with a fluorogenic peptide encompassing the of apoA-I by TTR. Abz-ESFKVS-EDDnp is an peptide in which is the and is the A of peptide substrate was by the peptide in (Sigma) to a final of TTR was to the substrate in an at a final of μl of reaction mm Tris, pH of the fluorogenic substrate was by at = and = in an The of the reaction was followed for at 37 into of a calibration was from the of the fluorogenic peptide with is also able to by of Abz-ESFKVS-EDDnp substrate with of in a final of 200 μl of reaction overnight at 37 from to of peptide substrate were at = and = was determined to using of substrate from to and 5 of TTR. The optimum pH for TTR activity was determined in 50 mm with pH from to TTR complexed with RBP was by in a for at 37 °C in the between TTR and was performed to (15Almeida M.R. Damas A.M. Lans M.C. Brower A. Saraiva M.J. Endocrine. 1997; 6: 309-315Crossref PubMed Scopus (85) Google Scholar). After TTR activity was as described a control for the effect of TTR activity was in the presence of RBP or under the conditions used for TTR. lines were in in and at 37 °C in a and were in with bovine serum 50 and 50 cell and cell lines were from the TTR and with high of TTR cell lines were by cell lines were with wt TTR in the which the M.M. Fernandes R. A. P. Saraiva M.J. Am. J. Pathol. Full Text Full Text PDF PubMed Scopus Google Scholar), and with a plasmid to by following the J. A Scholar). cell lines with wt TTR cDNA were and cell lines were with a carrying wt TTR cDNA under the control of the TTR a from of and the plasmid using to the Briefly, cells were in culture and to a of and plasmid for 5 after which with bovine serum was were in with (1 and cell lines with wt TTR cDNA were wt and wt, and cells were after which the culture was and by bovine serum with is an substrate with which is used as a substrate for several Proteolysis of the of the of proteolytic activity. mg/ml or were to the culture were on a 200 were with a coupled using or the = and = In using wt, wt, and wt cells were incubated with of Abz-ESFKVS-EDDnp at 37 °C for the effect of RBP, a of this TTR was to the culture for at 37 °C the addition of of proteolysis was performed by using μl of and at = and = in a After media, cells were with and protein was performed by the using bovine serum albumin from 50 to 200 as TTR in cell was by a TTR were overnight at °C with TTR in and with in μl of were for at was performed using TTR in phosphate and and TTR was from a from 5 to 200 activity was defined as of substrate of protein in cell were and a of TTR have previously that a fraction of plasma TTR circulates in through binding to apoA-I (4Sousa M.M. Berglund L. Saraiva M.J. J. Lipid Res. 2000; 41: 58-65Abstract Full Text Full Text PDF PubMed Google Scholar). further the nature of the interaction between TTR and apoA-I, we to a of the proteins by at 37 °C and physiological we that apoA-I was in the presence of recombinant TTR A major apoA-I fragment of kDa was however, with apoA-I fragments could be not This was the first evidence suggesting that TTR might be a cryptic protease able to apoA-I as a this of apoA-I with plasma TTR was and the of was The that of apoA-I with recombinant TTR or plasma TTR from different sources generated the of apoA-I to a proteolytic activity and against the presence of a protease in the TTR presented proteolytic activity or activity with a for protein in was performed in of the recombinant TTR and only TTR-related were not further suggesting that TTR is a novel cryptic further the of TTR to act as a we used is to act as a protease substrate M.C. D. PubMed Scopus Google Scholar). TTR was run on major band of was TTR was responsible for the of the we performed analysis of which that the band with activity in to TTR Protease by characterize the nature of the proteolytic activity of TTR, we used a of protease inhibitors and to apoA-I by TTR. an inhibitor of all of inhibited proteolysis of apoA-I not the serine protease inhibitors Pefabloc, PMSF, and apoA-I by TTR, whereas inhibitors of protease were effect and TPCK, inhibitors of chymotrypsin-like serine were also able to TTR activity data suggested that TTR has a chymotrypsin-like serine protease activity. of the on the site in apoA-I, we by N-terminal of apoA-I fragments generated with TTR. the identified terminus of the major proteolytic fragment of apoA-I was we that was occurring in the C terminus of the protein, and we the major apoA-I proteolytic fragment by A apoA-I was from amino acid residues to of the protein can be seen by of the of peptides from apoA-I and apoA-I by TTR by TTR between amino acid residues and of apoA-I, the peptide corresponding to the was in the analysis of apoA-I by TTR determine the the fragment of apoA-I by TTR was to C-terminal This analysis identified the site in apoA-I as after Phe-225. proteolytic a fluorogenic peptide corresponding to amino acid residues 223-228 of apoA-I which the that is by TTR, was analysis of peptides from apoA-I and in a of the Abz-ESFKVS-EDDnp by TTR in were used to the proteolytic of the Abz-ESFKVS-EDDnp fluorogenic of the recombinant of protein, as as the TTR isolated from the to the fluorogenic In an to a between proteolysis and pathological or physiological the used both wt or different TTR mutations to or to with as The following TTR were (i) the most to which is with the most of a variant to have a effect on the of the and and with the of the TTR activity was from not suggesting that the occurring on amyloidogenic variants of the protein or in not proteolytic activity. The optimum pH determined for TTR was the optimum pH was determined for of apoA-I not The reaction of the fluorogenic peptide a of 29 as determined by The proteolytic activity of TTR purified by HPLC was in of the fractions in activity using Abz-ESFKVS-EDDnp as The fractions corresponding to TTR with the fractions proteolytic activity Abz-ESFKVS-EDDnp further TTR-specific of the fluorogenic The of the major TTR ligands and on its proteolytic activity was we whereas RBP inhibited TTR only produced an decrease of TTR proteolysis a control for the effect of TTR ligands in its proteolytic the activity of in the presence of RBP or was The activity of was by the presence of RBP or therefore that the produced by ligands is of Proteolysis the of TTR to its proteolytic activity in we by the of by TTR with the by with high of TTR is an substrate of and several serine that a and sensitive of proteolytic activity. presented high of of from the of of a decrease in the to degrade this Furthermore, in the presence of Pefabloc, a protease inhibitor of TTR to degrade as by the of of from the in the addition of a protease inhibitor that we previously to be to TTR-mediated not the of to degrade of suggested in TTR was proteolytically We followed by in which was by the TTR to TTR-related proteolysis in of Abz-ESFKVS-EDDnp in different cell lines expressing TTR was and with the activity of control cell lines lacking TTR of the cell lines expressing TTR presented an of of the fluorogenic peptide when with that of the cell lacking TTR We therefore that TTR produced in cells of different sources is able to a peptide corresponding to the C terminus of that this in of Abz-ESFKVS-EDDnp is we the of RBP, a TTR that its proteolytic using the cell In the presence of RBP, expressing TTR the to degrade to the of cells TTR This further that TTR is a novel cryptic protease with physiological In this report we TTR as a novel cryptic protease and apoA-I as of its this point we the hypothesis that under physiological conditions TTR might have be the of to further the role of TTR activity. The presence of a in which be with TTR, is the data reported namely the of from different the high of of as by the of the activity with the TTR isolated by HPLC, and the of activity by TTR In to the different sources of TTR used in it is to that the occurring in TTR is the of the that is able to and to be to and of TTR from both protein and recombinant TTR a major corresponding to the of the protein; a corresponding to TTR, which is by with also be Y. M. A. H. K. M. Saraiva M.J. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar). the of further recombinant and TTR can be as cryptic plasma have been including plasma a that has been to have in its structure a cryptic serine I. J. Biochem. PubMed Scopus Google Scholar). and isolated were used to substrate and in physiological and pathological J. H. J. Protein Chem. 2000; PubMed Scopus Google Scholar). on the of TTR proteolytic activity by the serine protease inhibitors and as as its for a on and an optimum pH of we a chymotrypsin-like serine protease activity for TTR. The of TTR on apoA-I has with characterized chymotrypsin-like namely a chymotrypsin-like serine that in Google Scholar), and chymotrypsin-like including cell and and that have for a on and amino acid residues in and Y. L. D. K. K. H. 1985; PubMed Scopus Google Scholar). of the structure of TTR to a catalytic is to TTR has four and four analysis of the structure of the TTR only to a arrangement with a catalytic Damas when was performed at TTR the to therefore that is not the catalytic not the that TTR might be a serine as several have been identified G. A. Biochem. 1998; Full Text Full Text PDF PubMed Scopus Google for the protein a protein with a to the protease of and a catalytic C. J. 2000; PubMed Scopus Google Scholar) in which the plays the role of a and the of for TTR is a of four identical and it is that residues from different can to the catalytic is to that a that has been as a of is a that is only as a A. S. R. G. H. 1998; PubMed Scopus Google Scholar). the that in the proteolytic activity of TTR might be by a we the effect of TTR ligands in its activity. that the binding site of in the TTR not to the proteolytically site of the protein, when TTR is complexed to its activity is however, binding of RBP to TTR This can be by by RBP of the or by the that the amino acid residues on TTR that RBP play a role in The C-terminal of apoA-I is important in both lipid binding and K. D. 2000; PubMed Scopus Google it has been reported previously that the of residues cholesterol in cells Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is therefore that the of the C-terminal of apoA-I performed by TTR has an on lipid metabolism. The of the C terminus of apoA-I to proteolysis is in the I. L. R. A. L. Biochem. J. PubMed Scopus Google Scholar) and with the that the terminus of this protein is when with the C has been reported that cell a chymotrypsin-like the C terminus of apoA-I in HDL particles at M. G. G. L. J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar), the that is by TTR. In this it was that the of apoA-I in HDL particles to cholesterol therefore the that extracellular proteolysis of apoA-I is leading to the and of cells in The is for TTR of apoA-I after therefore the of TTR to apoA-I in HDL or HDL be We have previously identified an amyloidogenic variant of apoA-I in which analysis of fibrils of N-terminal fragments of mutated apoA-I and wt TTR (13de Sousa M.M. Vital C. Ostler D. Fernandes R. Pouget-Abadie J. Carles D. Saraiva M.J. Am. J. Pathol. 2000; 156: 1911-1917Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). we identified C-terminal of apoA-I by TTR can that this trigger apoA-I fibril formation. the role of TTR proteolysis in and under physiological conditions, namely the of apoA-I as a substrate of TTR, might have an not only in but also in to lipid metabolism. The evidence that TTR a cryptic protease in its structure is with activity in J. H. J. Protein Chem. 2000; PubMed Scopus Google Scholar), an important for the physiological of cryptic We and for the of recombinant TTR, the of plasma TTR and RBP, and the We are to for N-terminal and mass and to for C-terminal