Phytochmimy, Vol. 30, No. 9, pp. 2829-2833, Printed in Great Britain.
1991 0
0031-9422/w $3.00+0.00 1991 Per-on Press plc
THE COMPLETE AMINO ACID SEQUENCE OF THE MAJOR KUNITZ TRYPSIN INHIBITOR FROM THE SEEDS OF PROSOPSZS JULZFLORA ANDRE N. MONTE NEGREIROS,MARTA M. CARVALHO,*Jo& XAVIERFILHO,* ALEJANDROBLANCO-LABRA,~ PETERR. SHEWRY$and MICHAELRICHARDSON§]~ Departamento de Biuquimica, Universidade Federal do Rio Grande do Norte, 59075 Natal (RN), Brazil; *Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Cearh, 60001 Fortaleza (CE), Brazil; TCINVESTAV, Apartado Postal 629, Irapuato, Guanajuato, Mexico; $Long A&on Research Station, Bristol, U.K.; ~~~~rnent of Biological Sciences, University
of Durham, Science Laboratories, South Road, Durham DHl 3LE, U.K. (Received 25 January 1991) Key Word Imtex-Prosopsisjuiiftora; Leguminosae; Mimosoideae; seeds; mesquite; trypsin inhibitor; amino acid
sequence.
Abstract-The major inhibitor of trypsin in seeds of Proso~sis~uli~ora was purified by pr~ipitation with ~rnoni~ sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiomet~c ratio of I : 1, but had only weak activity against chymotrypsin and chid not inhibit human salivary or porcine pancreatic a-amylases. SDS-PAGE indicated that the inhibitor has a M, of ca 20000, and IEF-PAGE showed that the p1 is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (u chain) and 38 residues (B chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtiiisin/a-amylase inhibitors from cereals and the tastemodif~~g protein miraculin.
INTRODUCTION
Drought-resistant species of the xerophytic shrub Mesquite (Prosopsis spp.) are of widespread occurrence in the dry regions of Central and South America where they have been long recognised for their value in re-afforestation, wood-production, shade-provision, apiculture and as a food source for domestic animals. The Brazilian mesquite or algaroba (Prosopsis j~~~~ora) currently covers 100000 hectares of otherwise unproductive land in the northeastern states of Rio Grande do Norte and Ceaia. This re8ion has prolonged dry seasons and frequently experiences intense droughts. However production of the fruits and seeds of this species is little affected by these dry conditions. The fruit pods and seeds contain high levels of sucrose, a mannose/galactose polysaccharide and 10-14% protein [I-4]. Preliminary studies on the nut~tion~ values and digestibility of the seeds and of a flour and syrup prepared from the whole fruits have suggested that these products might be an acceptable emergency source of foodstuff for humans during famine periods caused by extremes of drought [2, 41. However little is known about the potentially anti-nut~tional components such as enzyme inhibitors in these fruits and seeds. Seeds of the Leguminosae are almost invariably rich sources of numerous protein enzyme inhibitors [S, 61. Weder [7] has reported that proteinase inhibitors
IlAuthor to whom correspondence
should be addressed.
were present in variable but often high amounts in all of 22 species of the subfamily Mimosoideae which were examined, however, Prosopsis spp. were not investigated. We are currently investi~ting the range of enzyme inhibitors found in the fruits of Prosopsis julijfora, and describe the purification, characterization and complete amino acid sequence of the major trypsin inhibitor present in the seeds. RESULTSAND DIscUssION
Preliminary experiments revealed that the bulk of the proteinase inhibitors present in the acetone-defatted flour of P. juli$ora seeds were extracted by 0.15 M NaCl. The su~rnatant obtained after ~nt~fu~ng such extracts was very viscous owing to the presence of large quantities of a soluble mannose/galactose polysaccharide. The addition of ammonium sulphate to give 80% saturation caused the precipitation of this polysaccharide and all of the inhibitory proteins, but the two were readily separated following centrifugation as the polysaccharide material floated on the surface of the supernatant. After dialysis against 0.05 M phosphate buffer at pH 7.6, the proteins precipitated by the 80% saturation with ammonium sulphate were applied to a column of DEAESepharose equilibrated in the same buffer. The majority of the proteinase inhibitors did not bind and were eiuted from the column in the first peak (data not shown). The material in the first peak was collected, dialysed against
2829
A. N. MONTE NEGREIROSer ai.
2830
120 Fraction number
Fig. 1. Chromatography of proteinase inhibitors from seeds of Prosopsis ju/iJora on CM-Sepharose. The unbound material (first peak) from the chromatography on DEAE-Sepharose was applied to a column (2.5 x 40 cm) of CM-Sepharose in 0.05 M Pi buffer pH 5.5 and eluted by a linear gradient of O-O.3 M NaCl(4W ml of each) in the same buffer. The flow rate was 6OmI hr-I. The solid line indicates absorption at 230 nm; the dotted line (-~--O--W--) shows the presence of trypsin inhibitory activity.
0.05 M phosphate
buffer pH 5.5 and applied to a column of CM-Sepharose equilibrated in the same buffer. All of the proteinase inhibitors were adsorbed on the CMSepharose and were eluted with a linear gradient of O-O.3 M NaCl (Fig. 1). Six peaks were found, all of which had trypsin inhibitory activity. The peaks 1-3 contained predominantly lower M, inhibitors (6000-1OOCO) of the Bowman-Birk type (Richardson ef al. unpublished results), whereas peaks 4-6 contained inhibitors of cu 20 000. The fractions in peak 6 were combined, dialysed against distilled water and Iyophili~d. This material was then subjected to reverse phase HPLC on a preparative Vydac Cl8 column which was eluted with a linear gradient of acetonitrile in 0.1% aqueous TFA (Fig. 2). The minor peaks which eluted at an acetonitrile concentration of 23-26% were traces of the low M, inhibitors. The major peak eluted at 40% acetonitrile was found to contain a single protein (TICM6) which was a potent inhibitor of trypsin. When TICM6 was examined by SDS-PAGE under non-reducing conditions it gave a single band with a M, of ca 20000, but under reducing conditions two additional bands were visible with M,s of cu 1.5000 and 5000. These results indicate that TICM6 is probably composed of two polypeptide chains linked by one or more disulphide bonds. This type of two-chain structure has been reported previously for the M, too00 Kunitz proteinase inhibitors found in seeds of Adenanthera pavonina 181, Acacia elata [9], and AIbiziu julibrissin [lo], all species belonging to the same subfamily (Mimosoideae) as P. juliJora. The isoelectric point (PI) of TICM6 was measured as 8.8 by IEF-PAGE, which is close to the values of 8.5 and 9.4 reported for two similar proteinase inhibitors from the seeds of Psophocarpus tetragonolobus [ll]. The inhibition of bovine trypsin and chymotrypsin at pH 8.0 by increasing levels of TICM6 is shown in Fig. 3.
With bovine trypsin the titration curve was linear and the inhibition was complete. The data show that 1 mol of TICM6 reacts stoichiometrically with 1 mol of trypsin to form a 1: 1 mol: mol inhibitor-enzyme complex. The titration data indicate that TICM6 was only a weak inhibitor of bovine chymotrypsin. No inhibition of either human salivary or porcine pancreatic a-amylases could be detected even when TICM6 was incubated in a five-fold molar excess with these enzymes. This spectrum of inhibitory activity is typical of many previously reported Kunitz type proteinase inhibitors from seeds of the Leguminosae [S-lo], but some species do also contain proteins capable of inhibiting chymotrypsin stoichiometritally in a 1: 1 molar ratio [9, 121. Further studies are in progress to determine whether any of the other Kunitz isoinhibitors present in peaks 4 and 5 from the CMSepharose column are strong inhibitors of chymotrypsin. Although the results of SDS-PAGE under reducing conditions indicate that TICM6 consists of two chains, N-terminal analysis by the DABITC/PITC microsequencing method revealed only one amino acid (Ser). The two-chain structure of TICM6 was confirmed by get fiItration on a column of Biogel P-30. The native protein gave a single peak with a M, of ca 2000, but foflowing S-reduction and ~rboxymethylation two peaks with absorption at 230 nm were obtained. The smaller polypeptide (the second peak to elute) had Ser as its N-terminal, but the Iarger protein (first peak) had no detectable N-terminal amino acid and was assumed to be blocked either with an acctyl group or a pyroglutamyl residue. The blocked residue was subsequently identified as pyroglutamic acid as treatment of the protein with the enzyme pyroglutamate aminopeptidase yielded a product susceptible to N-terminal analysis. The complete amino acid sequences of the two chains which comprise the inhibitor TICM6 are shown in Fig. 4, together with the detaits of the overlapping peptides from
283 I
Amino acid sequence of trypsin inhibitors
0
20
40 TIME
60
X0
(min)
Fig. 2. Reverse phase HPLC of material in peak 6 from CM-Sepharose column on a preparative column (22 mm x 25 cm) of Vydac C 18. The sample (40 mg) was injected in 1.5 ml of 6 M guanidine HCI in 0.1% aqueous TFA. The column was eluted with a linear gradient of acetonitrile (O-50%) in 0.1% aqueous TFA at a flow rate of 10 mlmin- ‘. The column temperature was 28”. The solid line indicates absorption at 216 nm. The asterisks indicate trypsin inhibitory activities.
30
Molar ratlo (InhIbItor:
enzyme)
Fig. 3. Inhibition of porcine trypsin, bovine chymotrypsin, human salivary and porcine pancreatic a-amylases by increasing amounts of TICM6 from seeds of Prosopsis juliflora. -0--, trypsin; --C chymotrypsin; - x - a-amylase (human salivary or porcine pancreatic).
which the sequences were deduced. The larger a-chain contains 137 amino acids, with minor examples of microheterogeneity occurring in positions 87,120 and 122. The smaller @hain contains 38 residues with no detectable microheterogeneity. The sequences of both chains were in good agreement with the results of amino acid analyses. The M, of TICM6 calculated from the sequences of the two chains is 19 783, which corresponds well with the estimates made by SDS-PAGE and gel filtration. The clear sequence similarities which exist between the Prosopsis julijIora inhibitor TICM6 and a number of
other Kunitz proteinase inhibitors from legume seeds [8, 11, 13, 143 and the cereal inhibitors of subtilisin and endogenous x-amylases [ 15, 163 are shown in Fig. 5. The 22 amino acid residues which are conserved in all seven of these proteins include the four cysteines which are involved in the two disulphide bridges. As might be expected the Prosopsis julijZora, TICM6 shows most homology (67%) with the protein from Adenanthera paoonina [8], a species belonging to the same subfamily Mimosoideae. The homology exhibited with the other inhibitors is between 36 and 42% for the legume proteins, and only ca 26% with the cereal types. It should be noted that the proteins shown in Fig. 5 also have some limited sequence similarities with cathepsin D inhibitors from potatoes [17, 183 and the taste-modifying protein miraculin from Richadella dulcifica [19]. The alignment of the two-chain inhibitors from Prosopsis and Adenanthera with the single-chain inhibitors from the other legumes tends to support the previous suggestions [8-lo] that the two chains result from the proteolytic cleavage of a susceptible bond in a singlechain precursor. Examination of the carboxyl terminal region of the Prosopsis TICM6 z chain and the aminoterminal region of the j? chain reveals that both are very hydrophilic in nature and might form an exposed loop in the supposed single-chain precursor which would be highly susceptible to proteolysis. However it is not clear why this cleavage should only occur in members of the more primitive subfamily Mimosoideae and not in the proteins from the Papilionideae or the Caesalpinoideae.
EXPERIMENTAL Seeds. Mature seeds of Prosopsis julij7oro (SW) DC. were
collected at Natal, Rio Grande do Norte. Brazil.
2832
A. N. MONTENEGREIROS et al.
1 10 20 30
1991 0
0031-9422/w $3.00+0.00 1991 Per-on Press plc
THE COMPLETE AMINO ACID SEQUENCE OF THE MAJOR KUNITZ TRYPSIN INHIBITOR FROM THE SEEDS OF PROSOPSZS JULZFLORA ANDRE N. MONTE NEGREIROS,MARTA M. CARVALHO,*Jo& XAVIERFILHO,* ALEJANDROBLANCO-LABRA,~ PETERR. SHEWRY$and MICHAELRICHARDSON§]~ Departamento de Biuquimica, Universidade Federal do Rio Grande do Norte, 59075 Natal (RN), Brazil; *Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Cearh, 60001 Fortaleza (CE), Brazil; TCINVESTAV, Apartado Postal 629, Irapuato, Guanajuato, Mexico; $Long A&on Research Station, Bristol, U.K.; ~~~~rnent of Biological Sciences, University
of Durham, Science Laboratories, South Road, Durham DHl 3LE, U.K. (Received 25 January 1991) Key Word Imtex-Prosopsisjuiiftora; Leguminosae; Mimosoideae; seeds; mesquite; trypsin inhibitor; amino acid
sequence.
Abstract-The major inhibitor of trypsin in seeds of Proso~sis~uli~ora was purified by pr~ipitation with ~rnoni~ sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiomet~c ratio of I : 1, but had only weak activity against chymotrypsin and chid not inhibit human salivary or porcine pancreatic a-amylases. SDS-PAGE indicated that the inhibitor has a M, of ca 20000, and IEF-PAGE showed that the p1 is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (u chain) and 38 residues (B chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtiiisin/a-amylase inhibitors from cereals and the tastemodif~~g protein miraculin.
INTRODUCTION
Drought-resistant species of the xerophytic shrub Mesquite (Prosopsis spp.) are of widespread occurrence in the dry regions of Central and South America where they have been long recognised for their value in re-afforestation, wood-production, shade-provision, apiculture and as a food source for domestic animals. The Brazilian mesquite or algaroba (Prosopsis j~~~~ora) currently covers 100000 hectares of otherwise unproductive land in the northeastern states of Rio Grande do Norte and Ceaia. This re8ion has prolonged dry seasons and frequently experiences intense droughts. However production of the fruits and seeds of this species is little affected by these dry conditions. The fruit pods and seeds contain high levels of sucrose, a mannose/galactose polysaccharide and 10-14% protein [I-4]. Preliminary studies on the nut~tion~ values and digestibility of the seeds and of a flour and syrup prepared from the whole fruits have suggested that these products might be an acceptable emergency source of foodstuff for humans during famine periods caused by extremes of drought [2, 41. However little is known about the potentially anti-nut~tional components such as enzyme inhibitors in these fruits and seeds. Seeds of the Leguminosae are almost invariably rich sources of numerous protein enzyme inhibitors [S, 61. Weder [7] has reported that proteinase inhibitors
IlAuthor to whom correspondence
should be addressed.
were present in variable but often high amounts in all of 22 species of the subfamily Mimosoideae which were examined, however, Prosopsis spp. were not investigated. We are currently investi~ting the range of enzyme inhibitors found in the fruits of Prosopsis julijfora, and describe the purification, characterization and complete amino acid sequence of the major trypsin inhibitor present in the seeds. RESULTSAND DIscUssION
Preliminary experiments revealed that the bulk of the proteinase inhibitors present in the acetone-defatted flour of P. juli$ora seeds were extracted by 0.15 M NaCl. The su~rnatant obtained after ~nt~fu~ng such extracts was very viscous owing to the presence of large quantities of a soluble mannose/galactose polysaccharide. The addition of ammonium sulphate to give 80% saturation caused the precipitation of this polysaccharide and all of the inhibitory proteins, but the two were readily separated following centrifugation as the polysaccharide material floated on the surface of the supernatant. After dialysis against 0.05 M phosphate buffer at pH 7.6, the proteins precipitated by the 80% saturation with ammonium sulphate were applied to a column of DEAESepharose equilibrated in the same buffer. The majority of the proteinase inhibitors did not bind and were eiuted from the column in the first peak (data not shown). The material in the first peak was collected, dialysed against
2829
A. N. MONTE NEGREIROSer ai.
2830
120 Fraction number
Fig. 1. Chromatography of proteinase inhibitors from seeds of Prosopsis ju/iJora on CM-Sepharose. The unbound material (first peak) from the chromatography on DEAE-Sepharose was applied to a column (2.5 x 40 cm) of CM-Sepharose in 0.05 M Pi buffer pH 5.5 and eluted by a linear gradient of O-O.3 M NaCl(4W ml of each) in the same buffer. The flow rate was 6OmI hr-I. The solid line indicates absorption at 230 nm; the dotted line (-~--O--W--) shows the presence of trypsin inhibitory activity.
0.05 M phosphate
buffer pH 5.5 and applied to a column of CM-Sepharose equilibrated in the same buffer. All of the proteinase inhibitors were adsorbed on the CMSepharose and were eluted with a linear gradient of O-O.3 M NaCl (Fig. 1). Six peaks were found, all of which had trypsin inhibitory activity. The peaks 1-3 contained predominantly lower M, inhibitors (6000-1OOCO) of the Bowman-Birk type (Richardson ef al. unpublished results), whereas peaks 4-6 contained inhibitors of cu 20 000. The fractions in peak 6 were combined, dialysed against distilled water and Iyophili~d. This material was then subjected to reverse phase HPLC on a preparative Vydac Cl8 column which was eluted with a linear gradient of acetonitrile in 0.1% aqueous TFA (Fig. 2). The minor peaks which eluted at an acetonitrile concentration of 23-26% were traces of the low M, inhibitors. The major peak eluted at 40% acetonitrile was found to contain a single protein (TICM6) which was a potent inhibitor of trypsin. When TICM6 was examined by SDS-PAGE under non-reducing conditions it gave a single band with a M, of ca 20000, but under reducing conditions two additional bands were visible with M,s of cu 1.5000 and 5000. These results indicate that TICM6 is probably composed of two polypeptide chains linked by one or more disulphide bonds. This type of two-chain structure has been reported previously for the M, too00 Kunitz proteinase inhibitors found in seeds of Adenanthera pavonina 181, Acacia elata [9], and AIbiziu julibrissin [lo], all species belonging to the same subfamily (Mimosoideae) as P. juliJora. The isoelectric point (PI) of TICM6 was measured as 8.8 by IEF-PAGE, which is close to the values of 8.5 and 9.4 reported for two similar proteinase inhibitors from the seeds of Psophocarpus tetragonolobus [ll]. The inhibition of bovine trypsin and chymotrypsin at pH 8.0 by increasing levels of TICM6 is shown in Fig. 3.
With bovine trypsin the titration curve was linear and the inhibition was complete. The data show that 1 mol of TICM6 reacts stoichiometrically with 1 mol of trypsin to form a 1: 1 mol: mol inhibitor-enzyme complex. The titration data indicate that TICM6 was only a weak inhibitor of bovine chymotrypsin. No inhibition of either human salivary or porcine pancreatic a-amylases could be detected even when TICM6 was incubated in a five-fold molar excess with these enzymes. This spectrum of inhibitory activity is typical of many previously reported Kunitz type proteinase inhibitors from seeds of the Leguminosae [S-lo], but some species do also contain proteins capable of inhibiting chymotrypsin stoichiometritally in a 1: 1 molar ratio [9, 121. Further studies are in progress to determine whether any of the other Kunitz isoinhibitors present in peaks 4 and 5 from the CMSepharose column are strong inhibitors of chymotrypsin. Although the results of SDS-PAGE under reducing conditions indicate that TICM6 consists of two chains, N-terminal analysis by the DABITC/PITC microsequencing method revealed only one amino acid (Ser). The two-chain structure of TICM6 was confirmed by get fiItration on a column of Biogel P-30. The native protein gave a single peak with a M, of ca 2000, but foflowing S-reduction and ~rboxymethylation two peaks with absorption at 230 nm were obtained. The smaller polypeptide (the second peak to elute) had Ser as its N-terminal, but the Iarger protein (first peak) had no detectable N-terminal amino acid and was assumed to be blocked either with an acctyl group or a pyroglutamyl residue. The blocked residue was subsequently identified as pyroglutamic acid as treatment of the protein with the enzyme pyroglutamate aminopeptidase yielded a product susceptible to N-terminal analysis. The complete amino acid sequences of the two chains which comprise the inhibitor TICM6 are shown in Fig. 4, together with the detaits of the overlapping peptides from
283 I
Amino acid sequence of trypsin inhibitors
0
20
40 TIME
60
X0
(min)
Fig. 2. Reverse phase HPLC of material in peak 6 from CM-Sepharose column on a preparative column (22 mm x 25 cm) of Vydac C 18. The sample (40 mg) was injected in 1.5 ml of 6 M guanidine HCI in 0.1% aqueous TFA. The column was eluted with a linear gradient of acetonitrile (O-50%) in 0.1% aqueous TFA at a flow rate of 10 mlmin- ‘. The column temperature was 28”. The solid line indicates absorption at 216 nm. The asterisks indicate trypsin inhibitory activities.
30
Molar ratlo (InhIbItor:
enzyme)
Fig. 3. Inhibition of porcine trypsin, bovine chymotrypsin, human salivary and porcine pancreatic a-amylases by increasing amounts of TICM6 from seeds of Prosopsis juliflora. -0--, trypsin; --C chymotrypsin; - x - a-amylase (human salivary or porcine pancreatic).
which the sequences were deduced. The larger a-chain contains 137 amino acids, with minor examples of microheterogeneity occurring in positions 87,120 and 122. The smaller @hain contains 38 residues with no detectable microheterogeneity. The sequences of both chains were in good agreement with the results of amino acid analyses. The M, of TICM6 calculated from the sequences of the two chains is 19 783, which corresponds well with the estimates made by SDS-PAGE and gel filtration. The clear sequence similarities which exist between the Prosopsis julijIora inhibitor TICM6 and a number of
other Kunitz proteinase inhibitors from legume seeds [8, 11, 13, 143 and the cereal inhibitors of subtilisin and endogenous x-amylases [ 15, 163 are shown in Fig. 5. The 22 amino acid residues which are conserved in all seven of these proteins include the four cysteines which are involved in the two disulphide bridges. As might be expected the Prosopsis julijZora, TICM6 shows most homology (67%) with the protein from Adenanthera paoonina [8], a species belonging to the same subfamily Mimosoideae. The homology exhibited with the other inhibitors is between 36 and 42% for the legume proteins, and only ca 26% with the cereal types. It should be noted that the proteins shown in Fig. 5 also have some limited sequence similarities with cathepsin D inhibitors from potatoes [17, 183 and the taste-modifying protein miraculin from Richadella dulcifica [19]. The alignment of the two-chain inhibitors from Prosopsis and Adenanthera with the single-chain inhibitors from the other legumes tends to support the previous suggestions [8-lo] that the two chains result from the proteolytic cleavage of a susceptible bond in a singlechain precursor. Examination of the carboxyl terminal region of the Prosopsis TICM6 z chain and the aminoterminal region of the j? chain reveals that both are very hydrophilic in nature and might form an exposed loop in the supposed single-chain precursor which would be highly susceptible to proteolysis. However it is not clear why this cleavage should only occur in members of the more primitive subfamily Mimosoideae and not in the proteins from the Papilionideae or the Caesalpinoideae.
EXPERIMENTAL Seeds. Mature seeds of Prosopsis julij7oro (SW) DC. were
collected at Natal, Rio Grande do Norte. Brazil.
2832
A. N. MONTENEGREIROS et al.
1 10 20 30
