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Light chain immunoglobulins as substrates for collagenase
Immunochemistry, 1975. Vol. 12. pp. 93-95. Pergamon Press. Printed in Great Britain
COMMUNICATIONS TO THE EDITORS L I G H T C H A I N I M M U N O G L O B U L I N S AS S U B S T R A T E S FOR COLLAGENASE* M.-A. C O L E T T I - P R E V I E R O , J.-C. C A V A D O R E Centre de Recberches de Biochimie Macromol~culaire (CNRS) and Groupe de Recherche U-67 (INSERM), BP 5051, 34033-Montpellier, France and C. T O N N E L L E Institut Pasteur, Service de Recherches sur les Prot~ines (Dr. Y. Manuel), 77, rue Pasteur, 69365 Lyon 7, France (Received 20 June 1974) Abstract--The proteolytic action of collagenase on immunoglobulin light chains has been studied by mean of quantitative determination of N-terminal amino acid residues. L 0~) and L (K) appear to be susceptible to the enzymatic action and this characteristic is a highly specific and general one. The consequences of this finding for structural studies are discussed.
Collagenase is a proteolytic enzyme with practically only one susceptible substrate, collagen. Investigations on the nature of its activity using synthetic polypeptides resembling collagen have shown a high specificity of the enzyme, limited to the sequence -Pro-X-Y-Pro-, with cleavage between X and Y, the highest rates having been observed when Y is Gly (Harper, 1972). Theoretically there is very little probability to find a sequence susceptible to cleavage in other proteins than collagen, but when proline content is relatively high this probability exists. This seems to be the case for immunoglobulin light chains (L), and we wish to present in this work their behaviour as collagenase substrates. EXPERIMENTAL Collagenase (batch number 53K427) was purchased from Worthington Corp., Freehold, N.J., and was essentially free of spurious proteolytic activity. Human immunoglobulin light chains (Bence Jones Ta (Tonnelle, 1973), Ve and Ge) were isolated from the urine of patient with multiple myeloma and purified by gel filtration on Sephadex G 100, followed by preparative liquid phase electrophoresis on an Elphor VAP 1 (160 mA, 2300V, in Tris/citric acid buffer, pH8'6). Immunodiffusion and immunoelectrophoresis using specific antisera showed Bence Jones Ta and Ve to be light chains type lambda, whereas Bence Jones Ge was type kappa, fl2Microglobulin was isolated from the urine of patients with renal tubular disease using the technique described by Bergghrd and Bearn (1968). All proteins yielded a single N-terminal amino acid residue by phenylisothiocyanate (Blomback et al., 1966) and by pivalyl chloride (Prota et al., 1971) methods, and a single Cterminal amino acid residue by hydrazinolysis (Blackburn, 1970). * Publication No. 109 from the Center and the Research Group. q~
Automatic amino acid analysis was performed using a Beckman 120B autoanalyzer and gas chromatography using a Perkin Elmer model 900 adapted to fit glass capillary columns: glass capillaries were drawn and coated as previously described (Cavadore et al., 1974). Extent of enzymatic proteolysis The extent of collagenase catalysed proteolysis was determined by following the liberation of new N-terminal residues. Aliquots of the reaction mixture were withdrawn at suitable intervals of time and N-terminals were analysed as phenylthiohydantoins (Roseau and Pantel, 1969) with regeneration, in favourable cases, of the amino acid residues by alkaline hydrolysis, and as pivalyl derivatives by quantitative gas liquid chromatography (Cavadore et al., 1974). The substrate concentration was 10- aM and enzyme concentration 10-SM; the pH, unless otherwise stated, was 8 (ethylmorpholine/acetic acid 0'l M buffer) and the temperature 30°C as a maximum. In fact, the rate of hydrolysis is enhanced by raising the temperature but at 37°C in some cases a precipitation was noticed which altered the reliability of the quantitative N-terminal determination.
RESULTS AND DISCUSSION Experiments on proteins with different Pro content and presumably with different sequences are summarised in Table 1. It appears that only L are susceptible to collagenase action, releasing new N-terminals: all other proteins tested as substrates gave negative results. The rate of cleavage plotted against the pH value depicts a bell shaped curve with a maximum around 8 (Fig. 1), as normally encountered in collagenase action. In Fig. 2 a typical gas chromatogram pattern is reported showing the appearance of new N-terminals after 60 min at 30°C when kappa chain Ge is submitted to collagenase action.
94
Communications to the Editors Table 1. Action ofcollagenase on different proteins as measured by the liberation of new N-terminal amino acids N terminal (residues/moles of protein)
Carboxymethylated protein
Liberated N-terminals (residues/moles of protein} 30 min 60 min
Lysozyme Myoglobin fie Microgiobulin Lambda chain Ta
Lys (1) Val (1) Ileu (1) Ser (1)
0 0 0 Leu (0.7) Ser (0.5)
Lambda chain Ve
Tyr (1)
Leu (0.7) Phe (0.9) Ser (0-4)
Kappa chain Ge
Glu (1)
Leu (0"6) Phe (1)
--
0 0 0 Leu (1.1) Ser (0.8) Ala (tr.) Val (tr.) Leu (1) Phe (1) Ser (0.8) Asp (tr.) Leu ( 1) Phe (1) Ala (tr.) Val (tr.)
0.9
i.s. olu --0.7 E
g - - 0 . 5 I1~
._..._j
,,,J
I
/
--
I
5
c
o
m
o~ "o
0"3
)he
I
7
I
8 pH
oo
I
tw
leu
9 ~glu
B
Fig. 1. Effect ofpH on the hydrolysis of L (2) Ta, catalyzed by coUagcnase. Experiments were carried out at 30°C, 40 rain: the liberated N-terminals were estimated by quantitative gas-liquid chromatography. From the evidence presented here it appears that only L are substrates for collagenase among the six protein tested. As for the exact nature of the sequence involved in the cleavage further studies are in progress to establish whether or not sequences different from collagen-type are concerned. Nevertheless/~2 microglobulin, whose structure (Peterson et aL 1972) is considered related to the constant regions of L (2) and L (•) does not show the same susceptibility to collagenas¢ attack. The proteolytic cleavage brought about by the enzyme on the substrate is very limited (2-3 bonds, Table 1) and this could be a very helpful tool for sequence studies of light chains. In fact, the possibility.of separately sequencing suitable fragments of a big polypeptide chain highly simplifies the strategy of the structural study using traditional procedures. Furthermore the production of a small number of
I
4O
I
3o
20 Time, mn
I
i
m
io
t
Ioo0 2O/mn
Fig. 2. Gas chromatography patterns of N-terminal amino acid residues of L (x) Ge by pivalylation. A: before enzymatic hydrolysis. B: after enzymatic hydrolysis, 30°C, 60 min. N-pivalyl amino acid methyl esters were separated on a glass capillary column (35 m x ff028 cm) coated with XE60 (5%)and FFAP (0"5%) in CH2C12. Nitrogen carrier; flow rate 1 ml/min; injector heater at 250°C. The I.S. (internal standard) is pivalyl Phe OEt.
Communications to the Editors large fragments from a protein may be a convenient route to complete the sequence by entirely automatic procedures in liquid (Edman and Begg, 1967; Niall, 1973) and solid phase (Laursen, 1971 ; Previero et al., 1973). The results obtained using collagenase on L constitute a precedent encouraging the research for the use of unconventional enzymes in the field of protein sequence determination. In order to reduce the number of peptide bonds cleaved, these enzymes should be chosen when they exhibit their specificity towards a particular sequence of amino acids rather than towards a particular kind of residue. The rapid and economical technique for quantitative determination of N-terminals in peptide mixtures, here employed, should facilitate research on the suitable unconventional enzymes for a given protein or family of proteins.
Acknowledgements--This work was partially supported by grants from D61egation G6n6rale h la Recherche Scientifique et Technique (No. 72 70009) and from Fondation pour la Recherche M6dicale Fran¢aise. The skilful technical assistance of P. Pantel is greatfully acknowledged.
95 REFERENCES
"
Bergg~trd I. and Bearn A. G. (1968) J. biol. Chem. 243, 4095. Blackburn S. (1970) Protein Sequence Determination: Methods and Techniques. Dekker, New York. Blomback B., Blomback M., Eeman P, and Hessel B. (1966) Biochim. biophys. Acta 115, 371. Cavadore J. C., Nota G., Prota G. and Previero A. (1974) Analyt. Biochem. 60, 608. Edman P. and Begg G. (1967) Eur. J. Biochem. 1, 80. Harper E. (1972) CoUagenase (Edited by Mandl I.), p. 19. Gordon & Breach, New York. Laursen R. A. (1973) FEBS-Letters 33, 135. Niall H. D. (1973) Methods in Enzymology (Edited by Hirs C. H, W. and Timasheff N. Vol. 25B, p. 942. Academic Press, New York. Peterson P. A., Cunningham B. A., Bergghrd I. and Edelman G. M. (1972) Proc. natn. Acad. Sci. U.S.A. 69, 1697. Previero A., Derancourt J., Coletti-Previero M.-A. and Laursen R. A. (1973) FEBS Lett. 33, 135. Prota G., Chioccara F. and Previero A. (1971) Biochimie 53, 51. Roseau G. and Pantel P. (1969) J. Chromatog. 44, 392. Tonnelle C. (1973) Biochem. biophys. Res. Commun. 55, 1112.
COMMUNICATIONS TO THE EDITORS L I G H T C H A I N I M M U N O G L O B U L I N S AS S U B S T R A T E S FOR COLLAGENASE* M.-A. C O L E T T I - P R E V I E R O , J.-C. C A V A D O R E Centre de Recberches de Biochimie Macromol~culaire (CNRS) and Groupe de Recherche U-67 (INSERM), BP 5051, 34033-Montpellier, France and C. T O N N E L L E Institut Pasteur, Service de Recherches sur les Prot~ines (Dr. Y. Manuel), 77, rue Pasteur, 69365 Lyon 7, France (Received 20 June 1974) Abstract--The proteolytic action of collagenase on immunoglobulin light chains has been studied by mean of quantitative determination of N-terminal amino acid residues. L 0~) and L (K) appear to be susceptible to the enzymatic action and this characteristic is a highly specific and general one. The consequences of this finding for structural studies are discussed.
Collagenase is a proteolytic enzyme with practically only one susceptible substrate, collagen. Investigations on the nature of its activity using synthetic polypeptides resembling collagen have shown a high specificity of the enzyme, limited to the sequence -Pro-X-Y-Pro-, with cleavage between X and Y, the highest rates having been observed when Y is Gly (Harper, 1972). Theoretically there is very little probability to find a sequence susceptible to cleavage in other proteins than collagen, but when proline content is relatively high this probability exists. This seems to be the case for immunoglobulin light chains (L), and we wish to present in this work their behaviour as collagenase substrates. EXPERIMENTAL Collagenase (batch number 53K427) was purchased from Worthington Corp., Freehold, N.J., and was essentially free of spurious proteolytic activity. Human immunoglobulin light chains (Bence Jones Ta (Tonnelle, 1973), Ve and Ge) were isolated from the urine of patient with multiple myeloma and purified by gel filtration on Sephadex G 100, followed by preparative liquid phase electrophoresis on an Elphor VAP 1 (160 mA, 2300V, in Tris/citric acid buffer, pH8'6). Immunodiffusion and immunoelectrophoresis using specific antisera showed Bence Jones Ta and Ve to be light chains type lambda, whereas Bence Jones Ge was type kappa, fl2Microglobulin was isolated from the urine of patients with renal tubular disease using the technique described by Bergghrd and Bearn (1968). All proteins yielded a single N-terminal amino acid residue by phenylisothiocyanate (Blomback et al., 1966) and by pivalyl chloride (Prota et al., 1971) methods, and a single Cterminal amino acid residue by hydrazinolysis (Blackburn, 1970). * Publication No. 109 from the Center and the Research Group. q~
Automatic amino acid analysis was performed using a Beckman 120B autoanalyzer and gas chromatography using a Perkin Elmer model 900 adapted to fit glass capillary columns: glass capillaries were drawn and coated as previously described (Cavadore et al., 1974). Extent of enzymatic proteolysis The extent of collagenase catalysed proteolysis was determined by following the liberation of new N-terminal residues. Aliquots of the reaction mixture were withdrawn at suitable intervals of time and N-terminals were analysed as phenylthiohydantoins (Roseau and Pantel, 1969) with regeneration, in favourable cases, of the amino acid residues by alkaline hydrolysis, and as pivalyl derivatives by quantitative gas liquid chromatography (Cavadore et al., 1974). The substrate concentration was 10- aM and enzyme concentration 10-SM; the pH, unless otherwise stated, was 8 (ethylmorpholine/acetic acid 0'l M buffer) and the temperature 30°C as a maximum. In fact, the rate of hydrolysis is enhanced by raising the temperature but at 37°C in some cases a precipitation was noticed which altered the reliability of the quantitative N-terminal determination.
RESULTS AND DISCUSSION Experiments on proteins with different Pro content and presumably with different sequences are summarised in Table 1. It appears that only L are susceptible to collagenase action, releasing new N-terminals: all other proteins tested as substrates gave negative results. The rate of cleavage plotted against the pH value depicts a bell shaped curve with a maximum around 8 (Fig. 1), as normally encountered in collagenase action. In Fig. 2 a typical gas chromatogram pattern is reported showing the appearance of new N-terminals after 60 min at 30°C when kappa chain Ge is submitted to collagenase action.
94
Communications to the Editors Table 1. Action ofcollagenase on different proteins as measured by the liberation of new N-terminal amino acids N terminal (residues/moles of protein)
Carboxymethylated protein
Liberated N-terminals (residues/moles of protein} 30 min 60 min
Lysozyme Myoglobin fie Microgiobulin Lambda chain Ta
Lys (1) Val (1) Ileu (1) Ser (1)
0 0 0 Leu (0.7) Ser (0.5)
Lambda chain Ve
Tyr (1)
Leu (0.7) Phe (0.9) Ser (0-4)
Kappa chain Ge
Glu (1)
Leu (0"6) Phe (1)
--
0 0 0 Leu (1.1) Ser (0.8) Ala (tr.) Val (tr.) Leu (1) Phe (1) Ser (0.8) Asp (tr.) Leu ( 1) Phe (1) Ala (tr.) Val (tr.)
0.9
i.s. olu --0.7 E
g - - 0 . 5 I1~
._..._j
,,,J
I
/
--
I
5
c
o
m
o~ "o
0"3
)he
I
7
I
8 pH
oo
I
tw
leu
9 ~glu
B
Fig. 1. Effect ofpH on the hydrolysis of L (2) Ta, catalyzed by coUagcnase. Experiments were carried out at 30°C, 40 rain: the liberated N-terminals were estimated by quantitative gas-liquid chromatography. From the evidence presented here it appears that only L are substrates for collagenase among the six protein tested. As for the exact nature of the sequence involved in the cleavage further studies are in progress to establish whether or not sequences different from collagen-type are concerned. Nevertheless/~2 microglobulin, whose structure (Peterson et aL 1972) is considered related to the constant regions of L (2) and L (•) does not show the same susceptibility to collagenas¢ attack. The proteolytic cleavage brought about by the enzyme on the substrate is very limited (2-3 bonds, Table 1) and this could be a very helpful tool for sequence studies of light chains. In fact, the possibility.of separately sequencing suitable fragments of a big polypeptide chain highly simplifies the strategy of the structural study using traditional procedures. Furthermore the production of a small number of
I
4O
I
3o
20 Time, mn
I
i
m
io
t
Ioo0 2O/mn
Fig. 2. Gas chromatography patterns of N-terminal amino acid residues of L (x) Ge by pivalylation. A: before enzymatic hydrolysis. B: after enzymatic hydrolysis, 30°C, 60 min. N-pivalyl amino acid methyl esters were separated on a glass capillary column (35 m x ff028 cm) coated with XE60 (5%)and FFAP (0"5%) in CH2C12. Nitrogen carrier; flow rate 1 ml/min; injector heater at 250°C. The I.S. (internal standard) is pivalyl Phe OEt.
Communications to the Editors large fragments from a protein may be a convenient route to complete the sequence by entirely automatic procedures in liquid (Edman and Begg, 1967; Niall, 1973) and solid phase (Laursen, 1971 ; Previero et al., 1973). The results obtained using collagenase on L constitute a precedent encouraging the research for the use of unconventional enzymes in the field of protein sequence determination. In order to reduce the number of peptide bonds cleaved, these enzymes should be chosen when they exhibit their specificity towards a particular sequence of amino acids rather than towards a particular kind of residue. The rapid and economical technique for quantitative determination of N-terminals in peptide mixtures, here employed, should facilitate research on the suitable unconventional enzymes for a given protein or family of proteins.
Acknowledgements--This work was partially supported by grants from D61egation G6n6rale h la Recherche Scientifique et Technique (No. 72 70009) and from Fondation pour la Recherche M6dicale Fran¢aise. The skilful technical assistance of P. Pantel is greatfully acknowledged.
95 REFERENCES
"
Bergg~trd I. and Bearn A. G. (1968) J. biol. Chem. 243, 4095. Blackburn S. (1970) Protein Sequence Determination: Methods and Techniques. Dekker, New York. Blomback B., Blomback M., Eeman P, and Hessel B. (1966) Biochim. biophys. Acta 115, 371. Cavadore J. C., Nota G., Prota G. and Previero A. (1974) Analyt. Biochem. 60, 608. Edman P. and Begg G. (1967) Eur. J. Biochem. 1, 80. Harper E. (1972) CoUagenase (Edited by Mandl I.), p. 19. Gordon & Breach, New York. Laursen R. A. (1973) FEBS-Letters 33, 135. Niall H. D. (1973) Methods in Enzymology (Edited by Hirs C. H, W. and Timasheff N. Vol. 25B, p. 942. Academic Press, New York. Peterson P. A., Cunningham B. A., Bergghrd I. and Edelman G. M. (1972) Proc. natn. Acad. Sci. U.S.A. 69, 1697. Previero A., Derancourt J., Coletti-Previero M.-A. and Laursen R. A. (1973) FEBS Lett. 33, 135. Prota G., Chioccara F. and Previero A. (1971) Biochimie 53, 51. Roseau G. and Pantel P. (1969) J. Chromatog. 44, 392. Tonnelle C. (1973) Biochem. biophys. Res. Commun. 55, 1112.