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The sequence of a cDNA encoding functional murine C1-inhibitor protein1
Biochimica et Biophysica Acta 1352 Ž1997. 156–160
Short sequence-paper
The sequence of a cDNA encoding functional murine C1-inhibitor protein 1 Jacqueline A. Russell, Keith Whaley, Shaun Heaphy
)
Department of Microbiology and Immunology, UniÕersity of Leicester, Medical School, UniÕersity Road, Leicester, LE1 9HN UK Received 30 January 1997; revised 20 March 1997; accepted 24 March 1997
Abstract The murine C1-inhibitor protein is 482 amino acids long. It consists of an N-terminal domain of 118 amino acids rich in proline and threonine and a serpin domain. The N-terminal domain has 39% identity with the corresponding regions of human and bovine C1 inhibitor. Keywords: C1 inhibitor; Serpin; Protein; Sequence; ŽMouse.
C1 inhibitor is a member of the serpin Žserine protease inhibitor. superfamily of protease inhibitors. It is the sole known regulator of the complement proteins C1r and C1s, and an important regulator of other serine proteases such as kallikrein, plasmin and factors XIIa and XIa. The gene for human C1-inhibitor protein encodes a mature protein of 478 amino acids with a predicted Mr of 52.9 kDa w1x. The protein is extensively glycosylated and has an apparent Mr of 105 kDa as deduced from SDS PAGE run under reducing conditions and 115 kDa when non-reduced. The mature protein comprises an extensively glycosylated amino terminal extension of about 113 amino acids, the function of which is unknown, and the serpin domain. Results obtained from neutronscattering experiments suggest that the serpin domain forms a globular head from which the amino terminal
) Corresponding author. Fax: q44 1162 525030; E-mail: [email protected] 1 The sequence data reported in this paper are available from EMBL under accession number Y10386.
domain extends as a rod-like appendage w2x. The serpin domain is homologous to other members of the superfamily and its structure has been modelled based on the crystal structure of other serpins w3x. Removal of the N-terminal domain does not affect the serpin function of C1 inhibitor, at least with respect to C1r and C1s using in vitro biochemical assays w4x. We recently sequenced a cDNA clone of mouse C1 inhibitor. A third sequence for bovine factor XIIa inhibitor, which inhibits C1s and is homologous to human C1 inhibitor w5x, has also been deposited in the SwissProt database ŽP50448.. We are therefore able to compare the protein sequences, particularly of the N-terminal domain for features suggesting functionality. Screening. About 500 000 plaques from a mouse liver cDNA library Ž lZAP, Stratagene. were lifted onto filters and screened. A random primed w 32 PxdCTP-labelled probe was produced using rediprime ŽAmersham.. The primer template was human C1-inhibitor cDNA w1x. Filters were prehybridised in 0.8 M NaCl, 20 mM PIPES ŽpH 6.5., 50% deionised formamide, 0.5% SDS. Probe was added to
0167-4781r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 0 5 6 - 0
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
the hybridisation solution Ž; 10 6 cpmrml. and hybridisation was carried out at 428C, overnight. The filters were washed twice for 10 min at 428C in 1 = SSC, 0.1% SDS. Two independent, positive clones were picked and subcloned into pBS by in vivo excision as described by Stratagene. Both clones were fully sequenced on each strand by primer walking using the PRISM ready reaction dye deoxy terminator cycle sequencing kit ŽPerkin Elmer.. These data were further confirmed by manual sequencing using Sequenase version 2.0 ŽUSB..
157
Both of these clones were found to be incomplete at the 5X end. 5X RACE with mouse liver MarathonReady cDNA ŽClonetech. was used to isolate two copies of the 5X end of the transcript. This allowed the design of primers to the 5X end of the gene for use in RT-PCR. Altogether 5 independent sequences were determined. Primer extension analysis, as previously described in w6x was then used to determine the full length of the 5X UTR. A Northern blot of mouse liver mRNA using the cDNA clone as a probe showed that the transcript
Fig. 1. Complete cDNA and derived protein sequence for mouse C1 inhibitor. The initiation codon, stop codon and polyadenylation signal are shaded.
158
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
was approximately 2.2 kb in length. The cDNA isolated here ŽFig. 1. consisted of a total of 1795 nucleotides which contained an open reading frame coding for 504 amino acids, predicted Mr 55.5 kDa, flanked by 5X and 3X UTRs of 70 and 213 nucleotides respectively. Confirmation of C1-inhibitor actiÕity. To confirm that the clones encoded mouse C1 inhibitor, the serpin domain of both mouse and human C1 inhibitor was expressed. The cDNA was cloned into the pET16b expression vector ŽNovagen. which produces N-terminal histidine tagged fusion proteins. The proteins were expressed using a rabbit reticulocyte transcription-translation kit Ž Promega. incorporating w 35 Sxmethionine and purified using Talon affinity resin ŽClonetech. as recommended by the manufacturers. The expression products were incubated with a pure preparation of human C1s for 3 h, 378C. The products were separated by SDS PAGE and visualised by fluorography according to manufacturers instructions ŽAmplify, Amersham. . Fig. 2 clearly shows that following incubation of human C1 inhibitor with human C1s, an SDS-stable enzyme-inhibitor complex Ž approximately 70 kDa. was formed comprising C1 inhibitor and the light chain of C1s Ž Mr 27 kDa. . In addition, some of the C1 inhibitor was cleaved, without forming the SDS-stable C1s-C1 inhibitor complexes, and appeared as a single band below the native C1 inhibitor. The murine expression product also formed both adducts. This is typical of a serpinserine protease interaction and confirmed that a functional murine C1-inhibitor cDNA had been successfully cloned and the protein expressed. C1-inhibitor protein. The average identity between murine, human and bovine C1 inhibitor is 72% at the nucleotide level and 65.1% at the amino acid level. The signal peptide, which is predicted to be 22 residues long for murine C1 inhibitor, is 92.4% identical between the three proteins. The amino terminal extension, which has no known function is only 39% identical, whereas the serpin domain is highly conserved with 71% identity between the three C1 inhibitors. The P1 residue Žamino acid 470., which is the major determinant of serpin specificity, is conserved as an arginine between all three C1 inhibitors. Other important residues within the serpin reactive loop are conserved, consistent with the observed inhibition of human C1s activity. Recently it has been
Fig. 2. Interaction of human C1s with 35S-labelled mouse Žleft. and human Žright. C1 inhibitor. C1s and C1 inhibitor were incubated together at 378C for 3 h. The reaction products were separated by SDS-PAGE Ž8% gels run under reducing conditions. and the position of the bands was detected by fluorography. In the absence of C1s Žy., mouse and human C1 inhibitor appear as a 43 kDa band. In the presence of C1s an SDS-stable C1srC1 inhibitor complex is detected Ž ; 70 kDa. which comprises C1 inhibitor and the light chain of C1s Ž27 kDa.. C1 inhibitor is also cleaved by C1s and this product can be seen as a band below the intact protein.
suggested that the residues Q452, Q453 and F455 in the distal hinge region of human C1 inhibitor contribute to a secondary binding site for C1s which is essential for the formation of SDS-stable C1s-C1-inhibitor complexes w7x. The R453Q substitution in murine C1 inhibitor suggests that the recognition site in C1s can accommodate the positively charged side chain of arginine. The four cysteine residues, which are involved in the formation of disulphide bonds in human C1 inhibitor, are conserved between the three proteins. Murine C1 inhibitor has an additional cysteine which cannot be involved in forming an intramolecular disulphide bond but which could form an intermolecular disulphide bond. N-linked carbohydrates are attached to asparagine residues within the consensus sequence asp-x-serrthr w8x. There are six of these consensus sites in human C1 inhibitor, all of which are known to be glycosylated. For murine and bovine C1 inhibitors, there are 6 and 4 potential N-glycosylation sites respectively and one of these sites aligns for all three proteins ŽFig. 3..
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
159
Fig. 3. Alignment of the protein sequences from mouse and human C1 inhibitor and bovine factor XIIa inhibitor. Identical residues are boxed, the signal peptide cleavage site is marked with an arrow, predicted N- and O-linked glycosylation sites are shaded, conserved cysteine residues are marked ^ and the P1 residue ).
The fact that the amino terminal domain of C1 inhibitor protein has been conserved Ž39%. suggests that it plays an important role in C1-inhibitor function. What this role is, is not known, although, as already shown it may not be directly involved with serpin-protease complex formation. The SwissProt database was searched for sequences similar to the three amino terminal domains. Identities of up to 20% were found. All the higher string matches were with protein domains containing multiple O-glycosylation sites, such as mucins. Serine and threonine residues are sites for O-glycosylation and where they are glycosylated there is typically an increased proportion of proline residues. Using a neural network possible targets for O-glycosylation were identified w9x. For murine C1 inhibitor a total of 15 O-glycosylation sites were predicted of which 13 were in the amino terminal domain. For human and bovine C1 inhibitor 26 of 32 and 5 of 8 predicted sites respectively lay in the amino terminal domain. This skewed distribution of predicted O-glycosylation reflects an increased proportion of threonine and proline residues in the extension domain. The role of glycosylation varies from one protein to another. O-glycosylation can influence protein
folding, secretion, conformational stability, proteolysis, antigenicity, plasma half-life, cell recognition and receptor activity w10x. It has been suggested that the amino terminal domain of human C1 inhibitor adopts an extended conformation w2x. Extensive O-glycosylation may stabilise this conformation as is the case for ovine submaxillary mucin w11x. It seems reasonable to assume that the N-terminal domains of murine and bovine C1 inhibitor form a similarly extended, Oglycosylation stabilised, conformation, the role of which requires further experimental investigation. It may modulate intermolecular interactions, possibly with target proteases including the C1 complex, surface bound C1q, other C1-inhibitor molecules or something else. We would like to thank the Wellcome Foundation for funding this work and the award of a studentship to J.A.R. Ž038487.. References w1x S.C. Bock, K. Skriver, E. Meilson, H.-C. Thogerson, B. Wiman, V.H. Donaldson, R.L. Eddy, J. Marriman, E. Radziejewska, R. Hunber, T.B. Shows, S. Magnusson, Biochemistry 25 Ž1986. 4292–4301.
160
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
w2x S.J. Perkins, K.F. Smith, J. Mol. Biol. 214 Ž1990. 751–763. w3x S. He, S. Tsang, J. North, N. Chohan, R.B. Sim, K. Whaley, J. Immunol. 156 Ž1996. 2009–2013. w4x M. Coutinho, K.S. Aulak, A.E. Davis III, J. Immunol. 153 Ž1994. 3648–3654. w5x M. Muldbjerg, S. Markussen, S. Magnusson, T. Halkier, Blood Coagul. Fibrinolysis 4 Ž1993. 47–54. w6x P.E. Carter, C. Duponchel, M. Tosi, J.E. Fothergill, Eur. J. Biochem. 197 Ž1991. 301–308. w7x S. He, R.B. Sim, K. Whaley, FEBS Lett. Ž19970 in press.
w8x H. Schachter, in: Glycoproteins, J. Montreuil, J.F.G. Vliegenhart, H. Schachter ŽEds.. Elsevier, Amsterdam, 1995, pp. 281-286. w9x J.E. Hansen, O. Lund, J. Engelbrecht, H. Bohr, J.O. Neilsen, J.E.S. Hansen, S. Brunak, Biochem. J. 308 Ž1995. 801–813. w10x I. Brockhausen, in: Glycoproteins, J. Montreuil, J.F.G. Vliegenhart, H. Schachter ŽEds.., Elsevier, Amsterdam, 1995, pp. 201-259, w11x R. Shogren, T.A. Gerken, N. Jentoft, Biochemistry 28 Ž1989. 5525–5536.
Short sequence-paper
The sequence of a cDNA encoding functional murine C1-inhibitor protein 1 Jacqueline A. Russell, Keith Whaley, Shaun Heaphy
)
Department of Microbiology and Immunology, UniÕersity of Leicester, Medical School, UniÕersity Road, Leicester, LE1 9HN UK Received 30 January 1997; revised 20 March 1997; accepted 24 March 1997
Abstract The murine C1-inhibitor protein is 482 amino acids long. It consists of an N-terminal domain of 118 amino acids rich in proline and threonine and a serpin domain. The N-terminal domain has 39% identity with the corresponding regions of human and bovine C1 inhibitor. Keywords: C1 inhibitor; Serpin; Protein; Sequence; ŽMouse.
C1 inhibitor is a member of the serpin Žserine protease inhibitor. superfamily of protease inhibitors. It is the sole known regulator of the complement proteins C1r and C1s, and an important regulator of other serine proteases such as kallikrein, plasmin and factors XIIa and XIa. The gene for human C1-inhibitor protein encodes a mature protein of 478 amino acids with a predicted Mr of 52.9 kDa w1x. The protein is extensively glycosylated and has an apparent Mr of 105 kDa as deduced from SDS PAGE run under reducing conditions and 115 kDa when non-reduced. The mature protein comprises an extensively glycosylated amino terminal extension of about 113 amino acids, the function of which is unknown, and the serpin domain. Results obtained from neutronscattering experiments suggest that the serpin domain forms a globular head from which the amino terminal
) Corresponding author. Fax: q44 1162 525030; E-mail: [email protected] 1 The sequence data reported in this paper are available from EMBL under accession number Y10386.
domain extends as a rod-like appendage w2x. The serpin domain is homologous to other members of the superfamily and its structure has been modelled based on the crystal structure of other serpins w3x. Removal of the N-terminal domain does not affect the serpin function of C1 inhibitor, at least with respect to C1r and C1s using in vitro biochemical assays w4x. We recently sequenced a cDNA clone of mouse C1 inhibitor. A third sequence for bovine factor XIIa inhibitor, which inhibits C1s and is homologous to human C1 inhibitor w5x, has also been deposited in the SwissProt database ŽP50448.. We are therefore able to compare the protein sequences, particularly of the N-terminal domain for features suggesting functionality. Screening. About 500 000 plaques from a mouse liver cDNA library Ž lZAP, Stratagene. were lifted onto filters and screened. A random primed w 32 PxdCTP-labelled probe was produced using rediprime ŽAmersham.. The primer template was human C1-inhibitor cDNA w1x. Filters were prehybridised in 0.8 M NaCl, 20 mM PIPES ŽpH 6.5., 50% deionised formamide, 0.5% SDS. Probe was added to
0167-4781r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 0 5 6 - 0
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
the hybridisation solution Ž; 10 6 cpmrml. and hybridisation was carried out at 428C, overnight. The filters were washed twice for 10 min at 428C in 1 = SSC, 0.1% SDS. Two independent, positive clones were picked and subcloned into pBS by in vivo excision as described by Stratagene. Both clones were fully sequenced on each strand by primer walking using the PRISM ready reaction dye deoxy terminator cycle sequencing kit ŽPerkin Elmer.. These data were further confirmed by manual sequencing using Sequenase version 2.0 ŽUSB..
157
Both of these clones were found to be incomplete at the 5X end. 5X RACE with mouse liver MarathonReady cDNA ŽClonetech. was used to isolate two copies of the 5X end of the transcript. This allowed the design of primers to the 5X end of the gene for use in RT-PCR. Altogether 5 independent sequences were determined. Primer extension analysis, as previously described in w6x was then used to determine the full length of the 5X UTR. A Northern blot of mouse liver mRNA using the cDNA clone as a probe showed that the transcript
Fig. 1. Complete cDNA and derived protein sequence for mouse C1 inhibitor. The initiation codon, stop codon and polyadenylation signal are shaded.
158
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
was approximately 2.2 kb in length. The cDNA isolated here ŽFig. 1. consisted of a total of 1795 nucleotides which contained an open reading frame coding for 504 amino acids, predicted Mr 55.5 kDa, flanked by 5X and 3X UTRs of 70 and 213 nucleotides respectively. Confirmation of C1-inhibitor actiÕity. To confirm that the clones encoded mouse C1 inhibitor, the serpin domain of both mouse and human C1 inhibitor was expressed. The cDNA was cloned into the pET16b expression vector ŽNovagen. which produces N-terminal histidine tagged fusion proteins. The proteins were expressed using a rabbit reticulocyte transcription-translation kit Ž Promega. incorporating w 35 Sxmethionine and purified using Talon affinity resin ŽClonetech. as recommended by the manufacturers. The expression products were incubated with a pure preparation of human C1s for 3 h, 378C. The products were separated by SDS PAGE and visualised by fluorography according to manufacturers instructions ŽAmplify, Amersham. . Fig. 2 clearly shows that following incubation of human C1 inhibitor with human C1s, an SDS-stable enzyme-inhibitor complex Ž approximately 70 kDa. was formed comprising C1 inhibitor and the light chain of C1s Ž Mr 27 kDa. . In addition, some of the C1 inhibitor was cleaved, without forming the SDS-stable C1s-C1 inhibitor complexes, and appeared as a single band below the native C1 inhibitor. The murine expression product also formed both adducts. This is typical of a serpinserine protease interaction and confirmed that a functional murine C1-inhibitor cDNA had been successfully cloned and the protein expressed. C1-inhibitor protein. The average identity between murine, human and bovine C1 inhibitor is 72% at the nucleotide level and 65.1% at the amino acid level. The signal peptide, which is predicted to be 22 residues long for murine C1 inhibitor, is 92.4% identical between the three proteins. The amino terminal extension, which has no known function is only 39% identical, whereas the serpin domain is highly conserved with 71% identity between the three C1 inhibitors. The P1 residue Žamino acid 470., which is the major determinant of serpin specificity, is conserved as an arginine between all three C1 inhibitors. Other important residues within the serpin reactive loop are conserved, consistent with the observed inhibition of human C1s activity. Recently it has been
Fig. 2. Interaction of human C1s with 35S-labelled mouse Žleft. and human Žright. C1 inhibitor. C1s and C1 inhibitor were incubated together at 378C for 3 h. The reaction products were separated by SDS-PAGE Ž8% gels run under reducing conditions. and the position of the bands was detected by fluorography. In the absence of C1s Žy., mouse and human C1 inhibitor appear as a 43 kDa band. In the presence of C1s an SDS-stable C1srC1 inhibitor complex is detected Ž ; 70 kDa. which comprises C1 inhibitor and the light chain of C1s Ž27 kDa.. C1 inhibitor is also cleaved by C1s and this product can be seen as a band below the intact protein.
suggested that the residues Q452, Q453 and F455 in the distal hinge region of human C1 inhibitor contribute to a secondary binding site for C1s which is essential for the formation of SDS-stable C1s-C1-inhibitor complexes w7x. The R453Q substitution in murine C1 inhibitor suggests that the recognition site in C1s can accommodate the positively charged side chain of arginine. The four cysteine residues, which are involved in the formation of disulphide bonds in human C1 inhibitor, are conserved between the three proteins. Murine C1 inhibitor has an additional cysteine which cannot be involved in forming an intramolecular disulphide bond but which could form an intermolecular disulphide bond. N-linked carbohydrates are attached to asparagine residues within the consensus sequence asp-x-serrthr w8x. There are six of these consensus sites in human C1 inhibitor, all of which are known to be glycosylated. For murine and bovine C1 inhibitors, there are 6 and 4 potential N-glycosylation sites respectively and one of these sites aligns for all three proteins ŽFig. 3..
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
159
Fig. 3. Alignment of the protein sequences from mouse and human C1 inhibitor and bovine factor XIIa inhibitor. Identical residues are boxed, the signal peptide cleavage site is marked with an arrow, predicted N- and O-linked glycosylation sites are shaded, conserved cysteine residues are marked ^ and the P1 residue ).
The fact that the amino terminal domain of C1 inhibitor protein has been conserved Ž39%. suggests that it plays an important role in C1-inhibitor function. What this role is, is not known, although, as already shown it may not be directly involved with serpin-protease complex formation. The SwissProt database was searched for sequences similar to the three amino terminal domains. Identities of up to 20% were found. All the higher string matches were with protein domains containing multiple O-glycosylation sites, such as mucins. Serine and threonine residues are sites for O-glycosylation and where they are glycosylated there is typically an increased proportion of proline residues. Using a neural network possible targets for O-glycosylation were identified w9x. For murine C1 inhibitor a total of 15 O-glycosylation sites were predicted of which 13 were in the amino terminal domain. For human and bovine C1 inhibitor 26 of 32 and 5 of 8 predicted sites respectively lay in the amino terminal domain. This skewed distribution of predicted O-glycosylation reflects an increased proportion of threonine and proline residues in the extension domain. The role of glycosylation varies from one protein to another. O-glycosylation can influence protein
folding, secretion, conformational stability, proteolysis, antigenicity, plasma half-life, cell recognition and receptor activity w10x. It has been suggested that the amino terminal domain of human C1 inhibitor adopts an extended conformation w2x. Extensive O-glycosylation may stabilise this conformation as is the case for ovine submaxillary mucin w11x. It seems reasonable to assume that the N-terminal domains of murine and bovine C1 inhibitor form a similarly extended, Oglycosylation stabilised, conformation, the role of which requires further experimental investigation. It may modulate intermolecular interactions, possibly with target proteases including the C1 complex, surface bound C1q, other C1-inhibitor molecules or something else. We would like to thank the Wellcome Foundation for funding this work and the award of a studentship to J.A.R. Ž038487.. References w1x S.C. Bock, K. Skriver, E. Meilson, H.-C. Thogerson, B. Wiman, V.H. Donaldson, R.L. Eddy, J. Marriman, E. Radziejewska, R. Hunber, T.B. Shows, S. Magnusson, Biochemistry 25 Ž1986. 4292–4301.
160
J.A. Russell et al.r Biochimica et Biophysica Acta 1352 (1997) 156–160
w2x S.J. Perkins, K.F. Smith, J. Mol. Biol. 214 Ž1990. 751–763. w3x S. He, S. Tsang, J. North, N. Chohan, R.B. Sim, K. Whaley, J. Immunol. 156 Ž1996. 2009–2013. w4x M. Coutinho, K.S. Aulak, A.E. Davis III, J. Immunol. 153 Ž1994. 3648–3654. w5x M. Muldbjerg, S. Markussen, S. Magnusson, T. Halkier, Blood Coagul. Fibrinolysis 4 Ž1993. 47–54. w6x P.E. Carter, C. Duponchel, M. Tosi, J.E. Fothergill, Eur. J. Biochem. 197 Ž1991. 301–308. w7x S. He, R.B. Sim, K. Whaley, FEBS Lett. Ž19970 in press.
w8x H. Schachter, in: Glycoproteins, J. Montreuil, J.F.G. Vliegenhart, H. Schachter ŽEds.. Elsevier, Amsterdam, 1995, pp. 281-286. w9x J.E. Hansen, O. Lund, J. Engelbrecht, H. Bohr, J.O. Neilsen, J.E.S. Hansen, S. Brunak, Biochem. J. 308 Ž1995. 801–813. w10x I. Brockhausen, in: Glycoproteins, J. Montreuil, J.F.G. Vliegenhart, H. Schachter ŽEds.., Elsevier, Amsterdam, 1995, pp. 201-259, w11x R. Shogren, T.A. Gerken, N. Jentoft, Biochemistry 28 Ž1989. 5525–5536.