Eur. J . Biochem. X2. 261 - 269 (1978)
The Primary Structure of Bovine Pancreatic Phospholipase A, Eduard A. M . FLEER, Hubertus M . VERHEIJ, and Gerard H. D E H A A S Biochemical Laboratory, State University of Utrecht (Received April 26, 1977)
The complete amino acid sequence of bovine phospholipase A, (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71 '0 of all residues of phospholipase A, from bovine, porcine and equine sources are conserved, bovine phospholipase A, differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions. Recently (pro)phospholipase A, has been isolated from several sources, including mammalian pancreas, snake and bee venom [l -61. The primary structure of a number of phospholipases has been elucidated and many authors have discussed the homology between venom and pancreatic phospholipases [5 - lo]. Studies on the mechanism of action have been reported for venom [l 1,121 and for pancreatic phospholipases [13,14]. Among the mammalian enzymes the bovine enzyme is clearly distinct from porcine enzyme with respect to pH optimum, Ca2+ binding, interaction with interfaces and specific activity [15- 181. Knowledge of the primary structure of this enzyme can improve our insight in the mechanism of action of phospholipases. The three-dimensional structure of porcine prophospholipase has been elucidated recently [ 191, but all attempts to obtain suitable crystals of the active form of this enzyme were unsuccessful. For this reason, only equine and bovine phospholipase A, crystals can be studied. High-resolution X-ray studies on the three-dimensional structure of active bovine phospholipase A, are in progress (Professor Drenth, personal communication). However, in order to interprete the X-ray density maps, knowledge of the primary
structure is essential, and we wish to report here the sequence of bovine phospholipase A,. MATERIALS AND METHODS Bovine phospholipase A, was isolated as previously described [2]. The following enzymes were used: trypsin, ~-l-tosylamido-2-phenylethylchloromethylketone-treated, was from Serva (Heidelberg); chymotrypsin, 3 times crystallized and 1-chloro-3-tosylamido-7-amino-2-heptanone-treatedbefore use, from Fluka (Switzerland); thermolysin, B-grade, was from Calbiochem (Los Angeles); staphylococcal protease from Miles (England); diaminopeptidyl hydrolase was isolated from beef spleen as described [20]; postproline cleaving enzyme [21] was a generous gift from Dr. R. Walter. Polyamide sheets were from Schleicher and Schull (Dassel, Germany): silicagel F256 thinlayer plates from Merck (Germany), and Sephadex G-50 fine and G-25 fine were obtained from Pharmacia (Sweden). Di-isopropylphosphorofluoridate was obtained from Mann's Laboratories and was used as a 0.1 M solution in anhydrous isopropanol. All other chemicals were of the highest available purity and were not further purified.
~~~
Ahhrevirrfions.Dansyl. 5-dimethylaminonaphthalene-1 -sulfonyl; Ruorescamine, 4-phenylspiro[furan-2(3H), 1 '-phthalan]-3,3'-dione. Enzymes. Iliaminopeptidylhydrolase (EC 3.4.14.1); chymotrypsin (EC 3.4.21.1); trypsin (EC 3.4.21.4); therrnolysin (EC 3.4.24.4); S t ~ t p h y k ~ ~ ooweus ~ ~ i i .external ~ protease (EC 3.4.99.-) ; carboxypeptidase A (EC 3.4.12.2).
Reduction of the Enzyme and Modijication of the Cysteine Residues
Unfolding and thialamination of the polypeptide chain was done as described before [8]. After lyophili-
262
zation, amino acid analysis showed that more than 97% of the cysteine residues were converted into thialaminine.
CNBr Digestion
To 50 mg thialaminated phospholipase A, (3 pmol) in 5 ml 70% formic acid was added 100 mg CNBr (1 mmol) dissolved in 5 ml 70% formic acid and the mixture was allowed to react for 24 h at 20 "C. After lyophilization, the peptides were separated by exclusion chromatography on a Sephadex G-50fine column.
Bovine Phospholipase A,
Secondurj, DigeslJ
Redigestion of the tryptic peptides T2 and T6 (0.5 pmol/ml in 1 mM NH4HC03)was accomplished with 0.5 mol% chymotrypsin for 30 min at 40 "C. Part of the peptide T2C4 (0.5 pmol/ml) was incubated with 0.5 mol% of thermolysin in 1 mM NH4HC03 for 20 h at 40 "C. The peptides T2C4 and CNBr, (1 .O pmol/ml) were incubated with 0.025 unit diaminopeptidyl hydrolase for 20 h at 40 "C using a pyridine/HCl buffer containing sodium chloride and 2-mercaptoethanol [20]. Separation of Peptides
Tryptic Digest
To 100 mg thialaminated phospholipase A, (6 pmol) dissolved in 10 ml 1 mM NH4HC03 0.5 mol% trypsin was added and the mixture was incubated for 15 min at 40 "C. Then 10 pmol di-isopropylphosphorofluoridate was added and after 5 min the solution was acidified with concentrated acetic acid to pH 3. After lyophilization the peptides were fractionated on a Sephadex G-50 fine column (200 x 3 cm), which was run in 0.1 M acetic acid. Peptides were detected at 206 and 280 nm, the fractions were pooled, lyophilized and further separated on a Sephadex G-25 fine column (200 x 3 cm). The peptides, obtained by this procedure, were purified further by high-voltage paper electrophoresis at pH 6.5 and descending paper chromatography as described before [8].
Digestion with Staphylococcal Protease
To 100 mg thialaminated protein (6 pmol) in 10 ml 1 mM NH4HC03, 0.5 mol% enzyme was added and the mixture was incubated for 2 h at 40 "C. Hereafter, I 0 pmol di-isopropylphosphorofluoridatewas added and allowed to react for 5 min. Peptides were isolated as described for the tryptic digest. The largest peptide of the digest (residues I - 59) was redigested with 1 mol% protease for 20 h at 40 "C in 1 mM NH4HC03and treated as described above.
Chymotryptic Digest
To 19mg thialaminated protein (1.2pmol)dissolved in 2 ml 0.1 M NH4HC0,, 0.5 mol% chymotrypsin (preincubated with l-chloro-3-tosylamido-7-amino-2heptanone) was added. The digestion was allowed to proceed for 1 h at 40 "C. Then 2 drops of acetic acid were added and the peptides were separated on a Sephadex G-50 column as described for the tryptic digest.
For analytical purposes peptide maps of suitable amounts of the peptide fractions were made, using high-voltage electrophoresis at pH 6.5 [8] and chromatography in either of the two solvent systems listed in Tables 1-3. After drying, the paper was sprayed with 10% (v/v) pyridine in acetone and followed with a solution of 2 mg fluorescamine in 100 ml 1% (v/v) pyridine in acetone. The fluorescent spots were cut out and the peptides were eluted with 0.1 M NH4HC03 (pH 9); 50% (v/v) pyridine and 50% (v/v) acetic acid, hydrolyzed and applied to the amino acid analyzer. Using this concentration of fluorescamine little or no loss of lysine and N-terminal residues occurs. From the results of these maps it was decided how a straightforward preparative purification could be carried out. Edman and Dansyl Procedure
The Edman procedure was the method described by Tarr [22]. In addition after each step dansylation was performed [23]. When peptides were sequenced through their penultimate residue, the COOH-terminal residue of the peptide chain was routinely determined on the amino acid analyzer. The phenylthiohydantoin derivatives were identified on thin-layer plates as described before [8]. If thialaminine, histidine or arginine were to be expected, subtractive amino acid analyses were performed too. Amino Acid Anulysis
Amino acid analyses were carried out on a Technicon TSM amino acid analyzer. Hydrolysis was for 24 h in 6 M HCl at 110 "C in carefully evacuated ampoules. Threonine and serine values are corrected for losses. Amide Assignment
The position of the amides in the sequence was determined by identification of the phenylthiohydantoin derivatives. In addition, the number of amides
263
E. A. M. Fleer, H. M. Verheij, and G. H. de Haas
present in the peptides was also determined by measuring electrophoretic mobility at pH 6.5 relative to aspartic acid [24]. Determination of Sulfhydryl Groups
starting with the first digestion, followed by a number after each letter to align them in the amino acid sequence. RESULTS
On the intact protein a determination of free sulfhydryl groups was carried out using dithiobisnitrobenzoic acid [25]. Peptide Nomenclature
The peptides obtained after CNBr cleavage are called CNBrl and CNBr2. Peptides obtained from enzymatic digestion are called T for tryptic peptides; C for chymotryptic peptides; G for staphylococcal protease peptides followed by numbers according to their alignment in the amino acid sequence. Peptides digested by more than one proteolytic enzyme are assigned T, C, and/or Th subsequently,
CNBr Cleavccge
CNBr cleavage resulted in two peptides: CNBr, , an octapeptide and CNBr, (1 15 amino acids, residues Ile9 - C Y S ' ~ CNBr, ~). was placed at the N-terminal of phospholipase, based on a comparison of its amino acid composition to the amino acid sequence of the N-terminal ofthe intact protein (Table 4). The sequence Am6-Gly7 in the polypeptide chain results in decrease in the yield of the Edman procedure, probably due to rearrangement [26]. We confirmed the sequence of the CNBr, peptide with diaminopeptidyl hydrolase digestion, which resulted in the peptides Ala-Leu ; Trp-Gln ; Phe-Asn and Gly-Hse.
Table 1. Amino acid coniposifion und charucterisrrcs of tryptic peprides Tryptophan was determined by ultraviolet spectroscopy. Purification methods : A, exclusion chromatography; B, high-voltage paper electrophoresis (50 V/cm; pH 6.5); C. descending paper chromatography in butan-1-ol/pyridine/acetic acid/H,O (15/10/3/12 by volume); D, as C in terr-butyl alcohoI!pyridine/formic acid/H,O (6/4/1/4 by volume). Values u are means of 4 hydrolysates. Hydrolysis times were 24 and 72 h. Values are corrected for losses. Values h are from the sequence Amino acid
PhospholipaseA, T, (1
h
109
11
T,
T,
T4
T,
T,
T,
T,
T,
T,,
10 25 10
10
17 13
11 09
19
10
46
09 10
1.9
1.0
~
Lysine Thialaminine Histidine Arginine Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Cysteine Valine Met hionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
09
-
20 20
1
I
2 2
25
25
4.0 10.0
4 10
8.0
8
5.2 6.0 5.8 13.3 3.6 1.0 4.7 8.1 7.0 4.0 0.9
5 6 6 14 4 1 5 8 7 4 1
10 30
08
09 0.9
1.1
1.3 1.2
09
6.4
2.1
0.9 3.0
1.0
1.0
1.0
Relative electrophoretic mobility ( p H 6 5)
123
-
9.9
1.0
4.6
0.8
2.7 4.1
1.1
0.9 3.1 2.1 1.2
1.0 0.9
1 .o
0.9
1.8
1.7
1.0
1 .o
1.6 1.1 2.8 1.0
0.8 0.9
1.o
0.7 1.2
t
10
33
025
N D
10
3
__ 6
068
OjO55
058
..~
38
N D
8
050
8
4
3
018
0
0 68
_-
~
Net charge at pH 6 5
-
-
+l
ND2+35
O/+l
+2
N D
+2
+07
0
+2 -~
~~~
Purification method ~
1.0
2.8
1.6
0.8
1.1 0.4 10 1.3
1.0
1.8
~
Number of residues
1.0
-
A.C
A,D
A,C
A,B,C
A,B.C
A,D
A,C
A,B,C
A,B.C
A,B,C
-
30
3s
65
3s
30
40
30
25
30
25
~~~~
Recovery
(O;)
264
Bovine Phospholipase A,
Table 2. Amino acid composition and characteristics of staphvlococral protease peptides Tryptophan was determined by ultraviolet spectroscopy. Purification methods: see Table 1. *Values are given for a mixture of two peptides CysW - Lys" and CysM - GlnS4 Amino acid Lysine Thialaminine Histidine Arginine Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine A 1an i n e Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan -
~
1.o 1 .o
1
~~
1
~
~
Recovery (",,,)
1.4
1.3
1 .o
5.9
1.o 4.7
2.0 3.0
2.0
4.2
1 .o
1 .o
1.o
1.1
2.9
1 .o
1.9 0.9 1 .o
1.7 1.9 1 .o
2.2
0.9 2.6
0.8
3.2
0.9
1.o
1.8
2.9 0.9 1 .o
1.o
1.o
1.o
0.9
1.o
0.8 2.0
1 -
~
~~
6
5
4
6
-
~~
-~
+1
0 -~
~
lO(11)
-~
-
N.D
0.56
N.D > + 3 . 5
f l
A.B,C
A.B,C
A,B.C
~
20
A,B,C ~~
20
9
Tryptic Digest
Tryptic digestion of the polypeptide chain resulted in 10 peptides which could be isolated in pure form (Table 1). The sites of cleavage are shown in Fig. 1. Despite the short incubation time and the use of 1.- 1 - tosylamido - 2 - phenylethylchloromethyl- ketonetreated trypsin partial cleavage between Trp3-Gln4 occurred (not indicated in Fig. 1). The peptides T3 up to TS were sequenced and the results are given in Table 4. Peptide T4 was isolated in two forms, peptides T4aand T4brespectively, both with the amino acid composition of Glx, Ala, Lys, but showing different electrophoretic mobilities : + 1 (T4a) and zero (T4b). This may be explained either by pyroglutamic acid formation or by deamidation. Since no N-terminal residue could be detected in T4b we ascribe this heterogeneity to rearrangement of the glutamine to pyroglutamic acid [27]. The peptides T2 (residues 11 -43) and Th (residues 63 - 100) were redigested with chymotrypsin. The resulting peptides are shown in Fig.2. Peptide T2C4 (residues 29 -43) could be sequenced up to its COOHterminal. The results of this Edman degradation were
25*
A.C __ 11
22
9
N.D
0.61
-
N.D
0
__ N.D
.-
0.36
~
5
0 -
~
~
15
6 -
~
-2
-.
~
22
5
-
~
4B.C ~
12
~.
~
A ~-
0.56
.~
+2
22 ~
0.63
0.45
0
Purification method ~
1.o 2.0
1.o
Net charge ~~
1 .o 3.7
1.1
~~~
~.
1.o 1.2
1.o
0.8 1 .o
1.1
1.2 1.o
1.9
1 .o
0.6 1.o
0.8
4.8
.o 1.o
1.o 1.8
1.9
2.9 1.o
1 .o
0.9
Relative electrophoretic mobility ~
0.9
2.0 1 .o
1
Number of residues -
1.2 1.1
1.1
A,B.C
A,C
A,B,C A.B.C __ 40 40 ~
65
30
A.C ~~
60
confirmed by redigesting T2C4 with thermolysin, which yielded the expected peptides (Fig. 2; Table 3). Peptide T2C2 was redigested with diaminopeptidyl hydrolase and the isolated dipeptides: Leu-Asp; PheAsn and Asn-Tyr confirmed the position and number of amides found in the peptides G,, and G,, (Fig. 1,2, Tables 2,3). The peptides T6Cz and T6C3 were sequenced (the results are shown in Table 4). Using the sequence of T6C3 the staphylococcal protease peptide G, could be aligned. Staphylococcal Protense Digestion Digestion with staphylococcal protease gave 5 peptides in good yields (G3 ; Fig. 1 ;Table 2). Peptide G2 was isolated in lower yield. The fraction called G, appeared to contain a mixture of two peptides: one containing residues 1 through 59 and one 5 residues shorter (corresponding to G2), thus explaining the lower yield of G 2 . When G, was further digested with the protease, several non-specific cleavages were observed, and peptides of suitable size for manual sequencing techniques were isolated in moderate yield.
~,
E. A. M. Fleer. H. M. Verheij. and G . H. de Haas
265
Table 3. Amino acid composition and charncterisrics of chymotryptir and thermol.vric peptides of'peptides T, and T6 and the chyrnotrjptic peptide C7 Tryptophan was determined by ultraviolet spectroscopy. Purification methods: see Table 1
Lysine Thialaminine Histidine Arginine Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Tryptophan
1 .o
2.9 1.o
0.9
0.9
4.8
0.8
I 2.1
}
3.1
2.0
1 .o 1 .o
1 .o
1
.o
2. I
2.1
5.8
1 .o
1.2 4.1
1.o
1.1
2.2 1.1
1
.o
1.o
1 .o
0.8
1 .o
9
~~~
__
~~
Relative electrophoretic mobility
0.24
_____
__
~
Net charge
+I ~~
Purification method Recovery ("J
1
.o
0.9 2.9
1 .o
0.9
0.9
2.0
1.9
1.o
I .o 4.2
1 .o
6
2.9
1.1
2.0
1.o
1.8
0.9
1 .o 1 .o
0.9 0.8
0.8 1
.o
__ 27 4
-~
__
Number of residues
~~
0.9
1.1
10
3.2
3
~~
6 ~
~
0.26
0
0.5
N.D
-1
0
+I ~
~
A,B,C A,B,C __ -~ 25 55
A,B,C
A.B.C
B,C
B, C
35
55
20
25
B. C
A,B,C
A,B,C
50
4s
~.
Among the observed splits are the ones on both sides of GlnS4, indicating that in staphylococcal protease peptides a COOH-terminal Glx is not necessarily Glu. That this partial splitting is not due to deamidation is evidenced by the finding that this peptide (Cys& - Gln54) could not be separated from CysU Lys5, by high-voltage electrophoresis and was further proven by carboxypeptidase A digestion of this mixture. The peptides G l , 4 ,GI., and G, to G, were sequenced successfully (Table 4). Chpmotryptic Digest Of the three chymotryptic fractions which were obtained after Sephadex gel filtration, 5 % of each was used for peptide maps from which amino acid analyses of the spots were performed after elution and hydrolysis. One of the fractions contained pure C, (Ala' -Trp3). The peptides were placed (Fig. 1 ) according to their amino acid composition and their relative electrophoretic mobility. Beside the main splits, minor peptides were detected which could be expected after cleavage at the COOH-terminus of Leu", Ile8* and Asn"' respectively.
20
0.96 ~-
~
N.D
+3 __ A,B.C A.B
-
~-
-
40
45
Peptide C7 was isolated preparatively for Edman/ dansyl degradation. This peptide showed a strong tendency to be washed out during sequencing, but when extraction after the coupling was carried out with hexane/ethyl acetate = 9/l (v/v), 5 successful Edman steps could be carried out leaving free leucine in 19U; yield as COOH-terminal residue. Sulfliydryl Group Determination Even in the presence of 8 M urea no free sulfhydryl groups could be detected in the intact protein. Therefore we conclude that all fourteen cysteine residues are present in disulfide bridges. Alignment of Peptides No problems were encountered in aligning the tryptie, staphylococcal and CNBr peptides except for peptides G4 and G, which could be interchanged (Fig. 1). Therefore, a chymotryptic digest of peptide Tb was done, which resulted among others in the isolation of peptide T6C3. Sequencing of this peptide clearly showed (Table 4) the position of G4, thus
266
Bovine Phospholipase A, d
d
-
a
A
d
10
d
20
A1 a -Leu -Trp -G1 n-Phe -Asn -G1 y-Met- I 1 e-Lys -Cys-Lys- I1 e-Pro-Ser-Ser-G1 u-Pro-Leu-Leu-Asp-Phe-Asn-Asn-Tyr I
I
’
1
I I
1 1
I
Ll
,
C?
fld:~]
I
m
. A 2A d - - - . -A_--.-‘
I 1
30
1
L
.I1
~
p
.
40
50
Gly-Cys-Tyr-Cys-Gly-Leu-Gly-Gly-Ser-Gly-Thr-Pro-Val-Asp-Asp-Leu-Asp-Ar~-Cys-Cys-Gl~-Thr-H~s-Asp-Asn -.---L-.-.---.
r
I 1
‘5
- A L L - -
I 1I
c1
‘1.t
.C
I -1 ’c 5
-
L
-7
#PI,>
70
60
Cys-Tyr-Lys-Gln-Ala-Lys-Lys-Leu-Asp-Ser-~-Lys-Val-Leu-Val-Asp-Asn-Pro-T~r-~hr-~sn-Asn-Tyr-Ser-Tyr 2
-
d
L A - -
I 1
1 1
\
4
d
2
-
.
-
.
d
1 1
4’ L? d
d
d
d
A
A
-
-
L
‘h
i
*I
4
1 1
i
A
-
-
-
b
d
d
d
-
-
.
-
L
d
I 1
d
-
.
s3
-
I 1
I I
I
L7
I 1
I 1
s
t
,fri.
I
i+
1
r,
9
90
&l’
Ser-Cys-Ser-Asn-Asn-Glu-Ile-Thr-Cys-Ser-Ser-Glu-Asn-Asn-Ala-Cy~-Glu-Ala-Phe-Ile-Cys-Asn-Cys-Asp-Ar~
P
t
.
.
.
i
T, ,
d
~
100
d
~
I I
2
-
-
~
d
-
-
1 1
b‘l
G5
(It,
Asn-Ala-~la-Ile-Cys-Phe-Ser-Lys-Val-Pro-Tyr-Asn-Lys-Glu-His-Lys-Asn-Leu-Asp-Lys-Lys-Asn-Cys
-------,
----
. I -
I ,
I 1
I 1
T7
T8
T ‘9 -.-L--L---L---
I
I
1 1
c7
7 ‘-13
c 15 t
Fig. I . Complete amino acid sequence oj’pho.vpho1ipa.w A, froin booine pancreas. Peptides obtained after various digests are indicated by lines. Partial splits are given by vertical dotted lines. -denotes sequence determined by Edman degradation
leaving only one position for G, . Between residues 52 and 59 overlap between tryptic and staphylococcal protease peptides was poor (Fig. 1). The isolation of the chymotryptic peptide C, confirmed the alignment unambiguously and moreover, the sequence of this peptide was in full agreement with the predicted structure (Table 4; Fig. 1).
DISCUSSION The post-proline cleaving enzyme, which was described recently [21], was thought useful for obtaining good overlaps. We incubated CNBr, , T2 and G, with this enzyme under conditions which would readily cleave tetrapeptides. No detectable degradation was observed with CNBr,, a slow degradation (10-20%
after 36 h digestion) was observed for T, and G,. Probably the use of this enzyme is mainly restricted to smaller peptides, an observation which was also made by Dr Walter (personal communication). The tryptic peptide T4 had glutamine as N-terminal residue, which partly cyclized to pyroglutamic acid. The conversion may be caused by the use of 0.1 M acetic acid during gel filtration, since it is known that this rearrangement is catalyzed by acidic conditions 1271. The active-center peptide T3 could be easily sequenced and no indication was found for a drop in the yield passing the glutamine residue. Working with active-center peptides from snake venom phospholipases, Joubert [9,28,29] reported this glutamine to be very sensitive to cyclization. Parallel to this study we also isolated the active-center peptide from porcine
~
~
E. A. M. Fleer, H. M. Verheij, and G. H. de Haas
261 20
Cys-Lys-Ile-Pro-Ser-Ser-Glu-Pro-Leu-Leu-Asp-Phe-Asn-Asn-Tyr-Gly-Cys-Tyr I 1 1
I
4-
TZC,
T, C
T2cz
30
2 3
40
Cys-Gly-Leu-Gly-Gly-Sey-Gly-ihy-Prp-Va!-Asp.-As~-Leu.-As~-Ar~ d
I
70
80
Val-Leu-Val-Asp-Asn-Pro-Tyr-Thr-Asn-Asn-Tyr-Ser-Tyr-S~r-Cys-Ser-Asn-Asn-Glu A
I
I I
Tbcl
~
2
~
l
~
1 1
'6'2
d
~
~
-
-
-
-
T6Cj
100
90
Ile-Thr-Cys-Ser-Ser-Glu-Asn-Asn-Ala-Cys-Glu-Ala-Phe-Ile-C~s-Asn-Cys-Asp-Arg 2 -
I
T6c3
Fig. 2. Secondary digests ofpeptides T2 and Ts with chymotrypsin (C) and digestion of T2C, with thermolysin ( T h ) . determined by Edman degradation
- denotes sequence
Table 4. Summary of sequence analysis of peptides derived from digests with proteolytic enzymes or cyanogen bromide of thialaminated phospholipase The residue position given is for the completed sequence (see Fig. 1). A denotes sequence determined by Edman degradation; 1indicates sites of attack of diaminopeptidyl hydrolase Peptide
Residue Position
Phospholipase A2
1-123
CNBrl
1-8
Number of Residues
Amino acid sequence (partially or complete)
123
Ala-Leu-Trp-Gln-Phe-Asn-(Gly,Met)-
8
1 - - r - - T 1 1 7
.L
1
1
Ala-Leu-Trp-Gln-Phe-Asn-Gly-H.Ser 22----2---L
CNBr2
9-123
115
Ile-Lys-Cys-Lys-Ile-Pro-Ser-Ser-Glu-Pro-Leu2 3 - 2 2 2
16-21
6
22-43
22
Phe-Asn-Asn-Tyr-G1y-Cys-Tyr-Cys-G1y-Leu -
29-43
15
Cys-Gly-Leu-Gly-Gly-Ser-Gly-Thr-Pro-Val-Asp-Asp-Leu-Asp-Arg
41-43
3
'3
44-53
10
c7
53-58
6
Lys-Gln-Ala-Lys-Lys-Leu
T4
54-56
3
G1 n-A1 a-Lys
T5
57-62
6
Lys-Leu-Asp-Ser-Cys-Lys
60-81
22
'6
63-100
38
T6C2
70-73
4
T6C3
74-100
27
G4
82-87
6
G5
88-92
5
G6
93-114
22
'7
101-108
a
T8
109-116
8
Ser-Glu-Pro-Leu-Leu-Asp 2 2 - - - - - - - - . l 2
- A - A - J L L - - - ~ L - ~ L 2
Leu-Asp-Arg L 2 - 2 - - . l 2 2 - -
G3
Cys-Cys-Gln-Thr-His-Asp-Asn-Cys-Tyr-Lys - - - 2 - - A -
----2
----+L2----
Ser-Cys-Lys-Val-Leu-Val-Asp-Asn-Pro-Tyr- - - \ L 2 - 2 - - - - - \ A 2 - -
Val-Leu-Val-Asp-Asn-Pro-Tyr-( A
L
L
)-Asn-Asn-Tyr-Ser-Tyr-
3
Thr-Asn-Asn-Tyr ---22------\-
Ser-Tyr-Ser-Cys-Ser-Asn-Asn-Glu-Ile-Thr2 2 L - 3 -
I1 e-Thr-Cys-Ser-Ser-G1u 2----
Asn-Asn-A1a-Cys-G1u L
2
3
L
A
L
l
L
3
-22--\-1
Asn-A1a-A1 a-I1 e-Cys-Phe-Ser-Lys - 3 - 2 - 3
G7
L15-123
9
2
L
L
Ala-Phe-Ile-Cys-Asn-Cys-Asp-Arg-Asn-Ala-Ala-Ile-
Val -Pro-Tyr-Asn-Lys-Glu-(His ,Lys) - 2 - 2 - . A L 2 2 - . J
His-Lys-Asn-Leu-Asp-Lys-Lys-Asn-Cys
268
Bovine Phospholipase A, 5
15
10
20
25
a. Ala-Leu-Trp-Gln-Phe-Asn-Gly-Met-Ile-Lys-Cys-Lys-Ile-Pro-Ser-Ser-Glu-Pro-Leu-Leu-Asp-Phe-Asn-Asn-Tyr b. . . . . . Arg-Ser . . Ala Gly His . . Yet . . . . c. Val . . . Arg-Ser . . Gin Thr . . Asn . Lys Tyr . Glu . . Asp
. .
.
. .
35
30
.
40
.
55
. .
65
60
.
50
45
Gly-Cys-Tyr-Cys-Gly-Leu-Gly-Gly-Ser-Gly-Thr-Pro-Val-Asp-Asp-Leu-Asp-Arg-Cys-Cys-Gln-Thr-His-Asp-Asn Glu . . . . Glu. . . . . . . . . . . . . . . . . Glu Aia Val . .
. . . . . . . . . . . . . .
.
.
. . .
.
70
75
Cys-Tyr-Lys-Gln-Ala-Lys-Lys-Leu-Asp-Ser-Cys-Lys-Val-Leu-Val-Asp-Asn-Pro-Tyr-Thr-Asn-Asn-Tyr-Ser-Tyr Arg-Asp . . Asn . . . . . Phe Glu-Ser . . . Thr Glu Ser Arg-Phe Glu-Ser Lys-Phe
. .
. .
. . .
.
. .
. . . . . . . . . . . . . .
85
80
90
.
95
100
Ser-Cys-Ser-Asn-Asn-Glu-Ile-Thr-Cys-Ser-Ser-Glu-Asn-Asn-Ala-Cys-Glu-Ala-Phe-Ile-Cys-Asn-Cys-Asp-Arg . . . . Thr. . . . Asn. Lys. . . . . . . . . . . . . . . Gly-Thr Val . . . Asp-Lys . . . . . . . . . . . .
.
105
110
115
120
-
. . 125
Asn-Ala-Ala-Ile-Cys-Phe-Ser-Lys-Ual-Pro-Tyr-Asn-Lys-Glu-His-Lys-Asn-Leu-Asp - Lys Lys-Asn-Cys . . . . . . . . Ala. . . . . . . . . . Thr. . Tyr . . . . . . . . . Ala . . . Pro Asn . . . . Ser Arg Ala
.
.
.
.
Fig. 3 . Amino m i d sequence comparison of bovine, porcine and equine pancreatic phospholipase A , . Sequences are: ( a ) bovine; (b) porcine and (c) equine. Points indicate sequence homology with the bovine sequence. Deletions ( - ) have been assigned to the bovine and porcine enzyme
phospholipase A,, which differs from the bovine active center peptide by a Lys/Arg and a Gln/Glu substitution [7]. When both peptides were sequenced at the same time under identical conditions, cyclization occurred to a large extent at glutamic acid and not at glutamine. Therefore, we must conclude that in certain peptides it is not glutamine but glutamic acid which has the strongest tendency to cyclicize. The redigestion of peptide G, with staphylococcal protease was carried out with another batch of enzyme than the one used for the first incubation. In this second digest more non-specific cleavages occurred, some of which have a trypsin-like character. The cleavage of the Lys"-thialaminine" and Arg43thialaminine@ both occurred in a strongly basic part of the polypeptide chain. This non-specific behaviour has been described before [30], but we did not try to investigate whether this non-specificity is due to the presence of autoproteolytic products or to the presence of traces of contaminating enzymes. In Fig. 3 the primary structures of bovine, porcine and equine phospholipases are shown. The homology between the bovine and porcine enzymes and between bovine and equine enzymes is 84% and 73% respectively. About 719{ of the polypeptide chain is conserved in all three species. The homology between snake venom and pancreatic phospholipases has been shown in several papers [ 5 - lo]; for reasons of spacesaving these data are not repeated here. However, it should be noted that the number of residues present in the COOH-terminal portion of the enzyme, which is variable for the mammalian species, is fairly constant in the elapid enzymes, whereas in both classes of enzymes this region carries a large excess of positive
charges. This basic cluster may be more important for enzymatic activity than the length of this external [19] loop. Another remarkable feature is the asparagine at position 6 in the bovine phospholipase A,, where arginine is found in the porcine and equine enzymes and lysine is found in most elapid enzymes. Perhaps the absence of a positive charge can be the cause of the lower activity and penetrating power of bovine phospholipase A2 as compared to porcine and equine enzymes (see below), since the N-terminal is of importance for substrate binding [31,32]. Since the homology between the mammalian phospholipases is rather high, a solution of the X-ray structure of the bovine enzyme can also be applied successfully to phospholipases from other sources. We wish to thank Drs A. M. Municio (Madrid) and C. E. Dutilh for their help in preliminary investigations, Mr W. C. Puijk for technical assistance in carrying out the amino acid analyses and D r H. Wang for carefully reading the manuscript. D r R. Walter kindly provided us with a sample of post-proline cleaving enzyme and synthetic substrate. This study was carried out in part under the auspices of The Netherlands Foundation of Chemical Research (S.O.N.) and with financial aid from The Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
REFERENCES 1. de Haas, G. H.. Postema, N . M., Nieuwenhuizen, W. & van Deenen, L. L. M. (1968) Biochim. Biophys. A d a , 159, 103-
-117 2. Dutilh, C. E., van Dam P. J., Verheul, F. E. A. M . & de Haas, G. H. (1975) Eur. J . Biochcwi. 53. 91 -97. 3. Evenberg, A., Meijer, H., Verheij, H. M. & de Haas. G. H. (1977) Biochim. Biophys. Acta. 491, 265 - 274.
269
E. A. M . Fleer. H. M . Verheij, and G. H. de Haas 4. Lowry, P. H.. Sarmiento, L. & Mitchell, H. K. (1971) Arch. Biochem. Biophys. 145. 338 - 343. 5. Halpert. J. & Eaker. D. (1975) J . Biol. Chem. 250, 6990-6997. 6. Tsao. F. H. C., Keim, P. S. & Heinrikson, R. L. (1975) Arch. Biochem. Biophys. 167, 706-717. 7. Puijk, W. C., Verheij. €I. M . & de Haas, G. 14. (1977) Bioc.hin1. Biopliys. Acta. 4Y2, 254- 259. 8. Evenberg, A,, Meijer, H.. Gaastra. W., Verheij, H. M . & de Haas, G. H. (1977) J . Biol. Chem. 252, 1189-1196. 9. Joubert. F. J. (1975) Eitr. J . Biochem. 52, 539 - 554. 10. Botes. D. P. and Viljoen, C. C. (1974) J . Biol. Chem. 249, 3827 3835. 11. Wells, M. A. (1971) Biochemistry. 10, 4074-4078. 12. Wells. M.A. (1973) Biochemisrry. 12, 1086-1093. 13. Slotboom. A. J., Pieterson, W. A,. Volwerk, J. J. & de Haas. G . H. (1976) Lipids (Paoletti, R., Bucellati, G. & Jacini. G.. eds) vol. I , pp. 99-107. Raven Press, New York. 14. Volwerk. J. J., Pieterson. W. A. & de Haas. G. H. (1974) Biochemisfry, 13, 1446- 1454. 15. Pieterson, W. A,, Vidal, J. C.. Volwerk, J. J . & de Haas. G. H. (1974) Biocheniistrv, 13, 1455- 1460. 16. de Haas, G. H.. Bonsen, P. P. M.. Pieterson, W. A . & van Deenen L. L. M. (1971) Biochim. Biophys. Acfa, 239, 252266 ~
17. Bonsen, P. P. M., de Haas, G. H., Pieterson. W. A. & van Deenen, L. L. M. (1972) Biochim. Biophys. Acta. 270. 364382. 18. Dutilh, C. E. (1976) Comparatice Studies on Phospholipase A , , Ph. D. Thesis, Utrecht, The Netherlands. 19. Drenth, J . , Enzing, C. M., Kalk, K. H. & Vessies. J. C. A . (1976) Nature (Lond.) 64, 373-377. 20. MacDonald. J. K., Callahan, P. X . & Ellis, S. (1972) Methods Enzymol. 256, 272 - 289. 21. Koida, M. & Walter, R. (1976) J . B i d . Chem. 251, 7593 - 7599. 22. Tarr, G. E. (1975) Anal. Biochem. 63. 361 -370. 23. Gray. W. R. (1972) Methods Enzymol. 25b. 333 - 334. 24. Otford, R. E. (1966) Nature (Lond.) 211. 591 -593. 25. Habeeb. A. F. S. A. (1972) Merhods En:vmo/. 25h, 458-459. 26. Koningsberg, W. (1972) Methods Enzymol. 25b. 326- 332. 27. Schroeder, W. A. (1972) Methods En:ymol. 25b, 307-313. 28. Joubert, F. J. (1975) Biochim. Biophys. Acta. 379, 329-344. 29. Joubert, F. J. (1975) Biochim. Biophys. Actu. 379. 345- 359. 30. Halpert, J. & Eaker, D. (1976) J . Biol. Chem. 251. 7343-7345. 31, van Wezel, F. & de Haas. G. H. (1975) Biochim. Biophys. Acta, 410. 299 - 309. 32. van Dam-Mieras. M. C. E., Slotboom, A . J., Pieterson, W. A. & dc Haas, G. H. (1974) Biochemistry. 14, 5387-5394.
E. A. M. Fleer. H. M. Verheij, and G. H. de Haas. Biochemisch Laboratorium. Rijksuniversiteit te Utrecht Transitorium 3. Universiteitscentrum "Lie Uithol”, Padualaan 8, Utrecht. The Netherlands
The Primary Structure of Bovine Pancreatic Phospholipase A, Eduard A. M . FLEER, Hubertus M . VERHEIJ, and Gerard H. D E H A A S Biochemical Laboratory, State University of Utrecht (Received April 26, 1977)
The complete amino acid sequence of bovine phospholipase A, (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71 '0 of all residues of phospholipase A, from bovine, porcine and equine sources are conserved, bovine phospholipase A, differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions. Recently (pro)phospholipase A, has been isolated from several sources, including mammalian pancreas, snake and bee venom [l -61. The primary structure of a number of phospholipases has been elucidated and many authors have discussed the homology between venom and pancreatic phospholipases [5 - lo]. Studies on the mechanism of action have been reported for venom [l 1,121 and for pancreatic phospholipases [13,14]. Among the mammalian enzymes the bovine enzyme is clearly distinct from porcine enzyme with respect to pH optimum, Ca2+ binding, interaction with interfaces and specific activity [15- 181. Knowledge of the primary structure of this enzyme can improve our insight in the mechanism of action of phospholipases. The three-dimensional structure of porcine prophospholipase has been elucidated recently [ 191, but all attempts to obtain suitable crystals of the active form of this enzyme were unsuccessful. For this reason, only equine and bovine phospholipase A, crystals can be studied. High-resolution X-ray studies on the three-dimensional structure of active bovine phospholipase A, are in progress (Professor Drenth, personal communication). However, in order to interprete the X-ray density maps, knowledge of the primary
structure is essential, and we wish to report here the sequence of bovine phospholipase A,. MATERIALS AND METHODS Bovine phospholipase A, was isolated as previously described [2]. The following enzymes were used: trypsin, ~-l-tosylamido-2-phenylethylchloromethylketone-treated, was from Serva (Heidelberg); chymotrypsin, 3 times crystallized and 1-chloro-3-tosylamido-7-amino-2-heptanone-treatedbefore use, from Fluka (Switzerland); thermolysin, B-grade, was from Calbiochem (Los Angeles); staphylococcal protease from Miles (England); diaminopeptidyl hydrolase was isolated from beef spleen as described [20]; postproline cleaving enzyme [21] was a generous gift from Dr. R. Walter. Polyamide sheets were from Schleicher and Schull (Dassel, Germany): silicagel F256 thinlayer plates from Merck (Germany), and Sephadex G-50 fine and G-25 fine were obtained from Pharmacia (Sweden). Di-isopropylphosphorofluoridate was obtained from Mann's Laboratories and was used as a 0.1 M solution in anhydrous isopropanol. All other chemicals were of the highest available purity and were not further purified.
~~~
Ahhrevirrfions.Dansyl. 5-dimethylaminonaphthalene-1 -sulfonyl; Ruorescamine, 4-phenylspiro[furan-2(3H), 1 '-phthalan]-3,3'-dione. Enzymes. Iliaminopeptidylhydrolase (EC 3.4.14.1); chymotrypsin (EC 3.4.21.1); trypsin (EC 3.4.21.4); therrnolysin (EC 3.4.24.4); S t ~ t p h y k ~ ~ ooweus ~ ~ i i .external ~ protease (EC 3.4.99.-) ; carboxypeptidase A (EC 3.4.12.2).
Reduction of the Enzyme and Modijication of the Cysteine Residues
Unfolding and thialamination of the polypeptide chain was done as described before [8]. After lyophili-
262
zation, amino acid analysis showed that more than 97% of the cysteine residues were converted into thialaminine.
CNBr Digestion
To 50 mg thialaminated phospholipase A, (3 pmol) in 5 ml 70% formic acid was added 100 mg CNBr (1 mmol) dissolved in 5 ml 70% formic acid and the mixture was allowed to react for 24 h at 20 "C. After lyophilization, the peptides were separated by exclusion chromatography on a Sephadex G-50fine column.
Bovine Phospholipase A,
Secondurj, DigeslJ
Redigestion of the tryptic peptides T2 and T6 (0.5 pmol/ml in 1 mM NH4HC03)was accomplished with 0.5 mol% chymotrypsin for 30 min at 40 "C. Part of the peptide T2C4 (0.5 pmol/ml) was incubated with 0.5 mol% of thermolysin in 1 mM NH4HC03 for 20 h at 40 "C. The peptides T2C4 and CNBr, (1 .O pmol/ml) were incubated with 0.025 unit diaminopeptidyl hydrolase for 20 h at 40 "C using a pyridine/HCl buffer containing sodium chloride and 2-mercaptoethanol [20]. Separation of Peptides
Tryptic Digest
To 100 mg thialaminated phospholipase A, (6 pmol) dissolved in 10 ml 1 mM NH4HC03 0.5 mol% trypsin was added and the mixture was incubated for 15 min at 40 "C. Then 10 pmol di-isopropylphosphorofluoridate was added and after 5 min the solution was acidified with concentrated acetic acid to pH 3. After lyophilization the peptides were fractionated on a Sephadex G-50 fine column (200 x 3 cm), which was run in 0.1 M acetic acid. Peptides were detected at 206 and 280 nm, the fractions were pooled, lyophilized and further separated on a Sephadex G-25 fine column (200 x 3 cm). The peptides, obtained by this procedure, were purified further by high-voltage paper electrophoresis at pH 6.5 and descending paper chromatography as described before [8].
Digestion with Staphylococcal Protease
To 100 mg thialaminated protein (6 pmol) in 10 ml 1 mM NH4HC03, 0.5 mol% enzyme was added and the mixture was incubated for 2 h at 40 "C. Hereafter, I 0 pmol di-isopropylphosphorofluoridatewas added and allowed to react for 5 min. Peptides were isolated as described for the tryptic digest. The largest peptide of the digest (residues I - 59) was redigested with 1 mol% protease for 20 h at 40 "C in 1 mM NH4HC03and treated as described above.
Chymotryptic Digest
To 19mg thialaminated protein (1.2pmol)dissolved in 2 ml 0.1 M NH4HC0,, 0.5 mol% chymotrypsin (preincubated with l-chloro-3-tosylamido-7-amino-2heptanone) was added. The digestion was allowed to proceed for 1 h at 40 "C. Then 2 drops of acetic acid were added and the peptides were separated on a Sephadex G-50 column as described for the tryptic digest.
For analytical purposes peptide maps of suitable amounts of the peptide fractions were made, using high-voltage electrophoresis at pH 6.5 [8] and chromatography in either of the two solvent systems listed in Tables 1-3. After drying, the paper was sprayed with 10% (v/v) pyridine in acetone and followed with a solution of 2 mg fluorescamine in 100 ml 1% (v/v) pyridine in acetone. The fluorescent spots were cut out and the peptides were eluted with 0.1 M NH4HC03 (pH 9); 50% (v/v) pyridine and 50% (v/v) acetic acid, hydrolyzed and applied to the amino acid analyzer. Using this concentration of fluorescamine little or no loss of lysine and N-terminal residues occurs. From the results of these maps it was decided how a straightforward preparative purification could be carried out. Edman and Dansyl Procedure
The Edman procedure was the method described by Tarr [22]. In addition after each step dansylation was performed [23]. When peptides were sequenced through their penultimate residue, the COOH-terminal residue of the peptide chain was routinely determined on the amino acid analyzer. The phenylthiohydantoin derivatives were identified on thin-layer plates as described before [8]. If thialaminine, histidine or arginine were to be expected, subtractive amino acid analyses were performed too. Amino Acid Anulysis
Amino acid analyses were carried out on a Technicon TSM amino acid analyzer. Hydrolysis was for 24 h in 6 M HCl at 110 "C in carefully evacuated ampoules. Threonine and serine values are corrected for losses. Amide Assignment
The position of the amides in the sequence was determined by identification of the phenylthiohydantoin derivatives. In addition, the number of amides
263
E. A. M. Fleer, H. M. Verheij, and G. H. de Haas
present in the peptides was also determined by measuring electrophoretic mobility at pH 6.5 relative to aspartic acid [24]. Determination of Sulfhydryl Groups
starting with the first digestion, followed by a number after each letter to align them in the amino acid sequence. RESULTS
On the intact protein a determination of free sulfhydryl groups was carried out using dithiobisnitrobenzoic acid [25]. Peptide Nomenclature
The peptides obtained after CNBr cleavage are called CNBrl and CNBr2. Peptides obtained from enzymatic digestion are called T for tryptic peptides; C for chymotryptic peptides; G for staphylococcal protease peptides followed by numbers according to their alignment in the amino acid sequence. Peptides digested by more than one proteolytic enzyme are assigned T, C, and/or Th subsequently,
CNBr Cleavccge
CNBr cleavage resulted in two peptides: CNBr, , an octapeptide and CNBr, (1 15 amino acids, residues Ile9 - C Y S ' ~ CNBr, ~). was placed at the N-terminal of phospholipase, based on a comparison of its amino acid composition to the amino acid sequence of the N-terminal ofthe intact protein (Table 4). The sequence Am6-Gly7 in the polypeptide chain results in decrease in the yield of the Edman procedure, probably due to rearrangement [26]. We confirmed the sequence of the CNBr, peptide with diaminopeptidyl hydrolase digestion, which resulted in the peptides Ala-Leu ; Trp-Gln ; Phe-Asn and Gly-Hse.
Table 1. Amino acid coniposifion und charucterisrrcs of tryptic peprides Tryptophan was determined by ultraviolet spectroscopy. Purification methods : A, exclusion chromatography; B, high-voltage paper electrophoresis (50 V/cm; pH 6.5); C. descending paper chromatography in butan-1-ol/pyridine/acetic acid/H,O (15/10/3/12 by volume); D, as C in terr-butyl alcohoI!pyridine/formic acid/H,O (6/4/1/4 by volume). Values u are means of 4 hydrolysates. Hydrolysis times were 24 and 72 h. Values are corrected for losses. Values h are from the sequence Amino acid
PhospholipaseA, T, (1
h
109
11
T,
T,
T4
T,
T,
T,
T,
T,
T,,
10 25 10
10
17 13
11 09
19
10
46
09 10
1.9
1.0
~
Lysine Thialaminine Histidine Arginine Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Cysteine Valine Met hionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
09
-
20 20
1
I
2 2
25
25
4.0 10.0
4 10
8.0
8
5.2 6.0 5.8 13.3 3.6 1.0 4.7 8.1 7.0 4.0 0.9
5 6 6 14 4 1 5 8 7 4 1
10 30
08
09 0.9
1.1
1.3 1.2
09
6.4
2.1
0.9 3.0
1.0
1.0
1.0
Relative electrophoretic mobility ( p H 6 5)
123
-
9.9
1.0
4.6
0.8
2.7 4.1
1.1
0.9 3.1 2.1 1.2
1.0 0.9
1 .o
0.9
1.8
1.7
1.0
1 .o
1.6 1.1 2.8 1.0
0.8 0.9
1.o
0.7 1.2
t
10
33
025
N D
10
3
__ 6
068
OjO55
058
..~
38
N D
8
050
8
4
3
018
0
0 68
_-
~
Net charge at pH 6 5
-
-
+l
ND2+35
O/+l
+2
N D
+2
+07
0
+2 -~
~~~
Purification method ~
1.0
2.8
1.6
0.8
1.1 0.4 10 1.3
1.0
1.8
~
Number of residues
1.0
-
A.C
A,D
A,C
A,B,C
A,B.C
A,D
A,C
A,B,C
A,B.C
A,B,C
-
30
3s
65
3s
30
40
30
25
30
25
~~~~
Recovery
(O;)
264
Bovine Phospholipase A,
Table 2. Amino acid composition and characteristics of staphvlococral protease peptides Tryptophan was determined by ultraviolet spectroscopy. Purification methods: see Table 1. *Values are given for a mixture of two peptides CysW - Lys" and CysM - GlnS4 Amino acid Lysine Thialaminine Histidine Arginine Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine A 1an i n e Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan -
~
1.o 1 .o
1
~~
1
~
~
Recovery (",,,)
1.4
1.3
1 .o
5.9
1.o 4.7
2.0 3.0
2.0
4.2
1 .o
1 .o
1.o
1.1
2.9
1 .o
1.9 0.9 1 .o
1.7 1.9 1 .o
2.2
0.9 2.6
0.8
3.2
0.9
1.o
1.8
2.9 0.9 1 .o
1.o
1.o
1.o
0.9
1.o
0.8 2.0
1 -
~
~~
6
5
4
6
-
~~
-~
+1
0 -~
~
lO(11)
-~
-
N.D
0.56
N.D > + 3 . 5
f l
A.B,C
A.B,C
A,B.C
~
20
A,B,C ~~
20
9
Tryptic Digest
Tryptic digestion of the polypeptide chain resulted in 10 peptides which could be isolated in pure form (Table 1). The sites of cleavage are shown in Fig. 1. Despite the short incubation time and the use of 1.- 1 - tosylamido - 2 - phenylethylchloromethyl- ketonetreated trypsin partial cleavage between Trp3-Gln4 occurred (not indicated in Fig. 1). The peptides T3 up to TS were sequenced and the results are given in Table 4. Peptide T4 was isolated in two forms, peptides T4aand T4brespectively, both with the amino acid composition of Glx, Ala, Lys, but showing different electrophoretic mobilities : + 1 (T4a) and zero (T4b). This may be explained either by pyroglutamic acid formation or by deamidation. Since no N-terminal residue could be detected in T4b we ascribe this heterogeneity to rearrangement of the glutamine to pyroglutamic acid [27]. The peptides T2 (residues 11 -43) and Th (residues 63 - 100) were redigested with chymotrypsin. The resulting peptides are shown in Fig.2. Peptide T2C4 (residues 29 -43) could be sequenced up to its COOHterminal. The results of this Edman degradation were
25*
A.C __ 11
22
9
N.D
0.61
-
N.D
0
__ N.D
.-
0.36
~
5
0 -
~
~
15
6 -
~
-2
-.
~
22
5
-
~
4B.C ~
12
~.
~
A ~-
0.56
.~
+2
22 ~
0.63
0.45
0
Purification method ~
1.o 2.0
1.o
Net charge ~~
1 .o 3.7
1.1
~~~
~.
1.o 1.2
1.o
0.8 1 .o
1.1
1.2 1.o
1.9
1 .o
0.6 1.o
0.8
4.8
.o 1.o
1.o 1.8
1.9
2.9 1.o
1 .o
0.9
Relative electrophoretic mobility ~
0.9
2.0 1 .o
1
Number of residues -
1.2 1.1
1.1
A,B.C
A,C
A,B,C A.B.C __ 40 40 ~
65
30
A.C ~~
60
confirmed by redigesting T2C4 with thermolysin, which yielded the expected peptides (Fig. 2; Table 3). Peptide T2C2 was redigested with diaminopeptidyl hydrolase and the isolated dipeptides: Leu-Asp; PheAsn and Asn-Tyr confirmed the position and number of amides found in the peptides G,, and G,, (Fig. 1,2, Tables 2,3). The peptides T6Cz and T6C3 were sequenced (the results are shown in Table 4). Using the sequence of T6C3 the staphylococcal protease peptide G, could be aligned. Staphylococcal Protense Digestion Digestion with staphylococcal protease gave 5 peptides in good yields (G3 ; Fig. 1 ;Table 2). Peptide G2 was isolated in lower yield. The fraction called G, appeared to contain a mixture of two peptides: one containing residues 1 through 59 and one 5 residues shorter (corresponding to G2), thus explaining the lower yield of G 2 . When G, was further digested with the protease, several non-specific cleavages were observed, and peptides of suitable size for manual sequencing techniques were isolated in moderate yield.
~,
E. A. M. Fleer. H. M. Verheij. and G . H. de Haas
265
Table 3. Amino acid composition and charncterisrics of chymotryptir and thermol.vric peptides of'peptides T, and T6 and the chyrnotrjptic peptide C7 Tryptophan was determined by ultraviolet spectroscopy. Purification methods: see Table 1
Lysine Thialaminine Histidine Arginine Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Tryptophan
1 .o
2.9 1.o
0.9
0.9
4.8
0.8
I 2.1
}
3.1
2.0
1 .o 1 .o
1 .o
1
.o
2. I
2.1
5.8
1 .o
1.2 4.1
1.o
1.1
2.2 1.1
1
.o
1.o
1 .o
0.8
1 .o
9
~~~
__
~~
Relative electrophoretic mobility
0.24
_____
__
~
Net charge
+I ~~
Purification method Recovery ("J
1
.o
0.9 2.9
1 .o
0.9
0.9
2.0
1.9
1.o
I .o 4.2
1 .o
6
2.9
1.1
2.0
1.o
1.8
0.9
1 .o 1 .o
0.9 0.8
0.8 1
.o
__ 27 4
-~
__
Number of residues
~~
0.9
1.1
10
3.2
3
~~
6 ~
~
0.26
0
0.5
N.D
-1
0
+I ~
~
A,B,C A,B,C __ -~ 25 55
A,B,C
A.B.C
B,C
B, C
35
55
20
25
B. C
A,B,C
A,B,C
50
4s
~.
Among the observed splits are the ones on both sides of GlnS4, indicating that in staphylococcal protease peptides a COOH-terminal Glx is not necessarily Glu. That this partial splitting is not due to deamidation is evidenced by the finding that this peptide (Cys& - Gln54) could not be separated from CysU Lys5, by high-voltage electrophoresis and was further proven by carboxypeptidase A digestion of this mixture. The peptides G l , 4 ,GI., and G, to G, were sequenced successfully (Table 4). Chpmotryptic Digest Of the three chymotryptic fractions which were obtained after Sephadex gel filtration, 5 % of each was used for peptide maps from which amino acid analyses of the spots were performed after elution and hydrolysis. One of the fractions contained pure C, (Ala' -Trp3). The peptides were placed (Fig. 1 ) according to their amino acid composition and their relative electrophoretic mobility. Beside the main splits, minor peptides were detected which could be expected after cleavage at the COOH-terminus of Leu", Ile8* and Asn"' respectively.
20
0.96 ~-
~
N.D
+3 __ A,B.C A.B
-
~-
-
40
45
Peptide C7 was isolated preparatively for Edman/ dansyl degradation. This peptide showed a strong tendency to be washed out during sequencing, but when extraction after the coupling was carried out with hexane/ethyl acetate = 9/l (v/v), 5 successful Edman steps could be carried out leaving free leucine in 19U; yield as COOH-terminal residue. Sulfliydryl Group Determination Even in the presence of 8 M urea no free sulfhydryl groups could be detected in the intact protein. Therefore we conclude that all fourteen cysteine residues are present in disulfide bridges. Alignment of Peptides No problems were encountered in aligning the tryptie, staphylococcal and CNBr peptides except for peptides G4 and G, which could be interchanged (Fig. 1). Therefore, a chymotryptic digest of peptide Tb was done, which resulted among others in the isolation of peptide T6C3. Sequencing of this peptide clearly showed (Table 4) the position of G4, thus
266
Bovine Phospholipase A, d
d
-
a
A
d
10
d
20
A1 a -Leu -Trp -G1 n-Phe -Asn -G1 y-Met- I 1 e-Lys -Cys-Lys- I1 e-Pro-Ser-Ser-G1 u-Pro-Leu-Leu-Asp-Phe-Asn-Asn-Tyr I
I
’
1
I I
1 1
I
Ll
,
C?
fld:~]
I
m
. A 2A d - - - . -A_--.-‘
I 1
30
1
L
.I1
~
p
.
40
50
Gly-Cys-Tyr-Cys-Gly-Leu-Gly-Gly-Ser-Gly-Thr-Pro-Val-Asp-Asp-Leu-Asp-Ar~-Cys-Cys-Gl~-Thr-H~s-Asp-Asn -.---L-.-.---.
r
I 1
‘5
- A L L - -
I 1I
c1
‘1.t
.C
I -1 ’c 5
-
L
-7
#PI,>
70
60
Cys-Tyr-Lys-Gln-Ala-Lys-Lys-Leu-Asp-Ser-~-Lys-Val-Leu-Val-Asp-Asn-Pro-T~r-~hr-~sn-Asn-Tyr-Ser-Tyr 2
-
d
L A - -
I 1
1 1
\
4
d
2
-
.
-
.
d
1 1
4’ L? d
d
d
d
A
A
-
-
L
‘h
i
*I
4
1 1
i
A
-
-
-
b
d
d
d
-
-
.
-
L
d
I 1
d
-
.
s3
-
I 1
I I
I
L7
I 1
I 1
s
t
,fri.
I
i+
1
r,
9
90
&l’
Ser-Cys-Ser-Asn-Asn-Glu-Ile-Thr-Cys-Ser-Ser-Glu-Asn-Asn-Ala-Cy~-Glu-Ala-Phe-Ile-Cys-Asn-Cys-Asp-Ar~
P
t
.
.
.
i
T, ,
d
~
100
d
~
I I
2
-
-
~
d
-
-
1 1
b‘l
G5
(It,
Asn-Ala-~la-Ile-Cys-Phe-Ser-Lys-Val-Pro-Tyr-Asn-Lys-Glu-His-Lys-Asn-Leu-Asp-Lys-Lys-Asn-Cys
-------,
----
. I -
I ,
I 1
I 1
T7
T8
T ‘9 -.-L--L---L---
I
I
1 1
c7
7 ‘-13
c 15 t
Fig. I . Complete amino acid sequence oj’pho.vpho1ipa.w A, froin booine pancreas. Peptides obtained after various digests are indicated by lines. Partial splits are given by vertical dotted lines. -denotes sequence determined by Edman degradation
leaving only one position for G, . Between residues 52 and 59 overlap between tryptic and staphylococcal protease peptides was poor (Fig. 1). The isolation of the chymotryptic peptide C, confirmed the alignment unambiguously and moreover, the sequence of this peptide was in full agreement with the predicted structure (Table 4; Fig. 1).
DISCUSSION The post-proline cleaving enzyme, which was described recently [21], was thought useful for obtaining good overlaps. We incubated CNBr, , T2 and G, with this enzyme under conditions which would readily cleave tetrapeptides. No detectable degradation was observed with CNBr,, a slow degradation (10-20%
after 36 h digestion) was observed for T, and G,. Probably the use of this enzyme is mainly restricted to smaller peptides, an observation which was also made by Dr Walter (personal communication). The tryptic peptide T4 had glutamine as N-terminal residue, which partly cyclized to pyroglutamic acid. The conversion may be caused by the use of 0.1 M acetic acid during gel filtration, since it is known that this rearrangement is catalyzed by acidic conditions 1271. The active-center peptide T3 could be easily sequenced and no indication was found for a drop in the yield passing the glutamine residue. Working with active-center peptides from snake venom phospholipases, Joubert [9,28,29] reported this glutamine to be very sensitive to cyclization. Parallel to this study we also isolated the active-center peptide from porcine
~
~
E. A. M. Fleer, H. M. Verheij, and G. H. de Haas
261 20
Cys-Lys-Ile-Pro-Ser-Ser-Glu-Pro-Leu-Leu-Asp-Phe-Asn-Asn-Tyr-Gly-Cys-Tyr I 1 1
I
4-
TZC,
T, C
T2cz
30
2 3
40
Cys-Gly-Leu-Gly-Gly-Sey-Gly-ihy-Prp-Va!-Asp.-As~-Leu.-As~-Ar~ d
I
70
80
Val-Leu-Val-Asp-Asn-Pro-Tyr-Thr-Asn-Asn-Tyr-Ser-Tyr-S~r-Cys-Ser-Asn-Asn-Glu A
I
I I
Tbcl
~
2
~
l
~
1 1
'6'2
d
~
~
-
-
-
-
T6Cj
100
90
Ile-Thr-Cys-Ser-Ser-Glu-Asn-Asn-Ala-Cys-Glu-Ala-Phe-Ile-C~s-Asn-Cys-Asp-Arg 2 -
I
T6c3
Fig. 2. Secondary digests ofpeptides T2 and Ts with chymotrypsin (C) and digestion of T2C, with thermolysin ( T h ) . determined by Edman degradation
- denotes sequence
Table 4. Summary of sequence analysis of peptides derived from digests with proteolytic enzymes or cyanogen bromide of thialaminated phospholipase The residue position given is for the completed sequence (see Fig. 1). A denotes sequence determined by Edman degradation; 1indicates sites of attack of diaminopeptidyl hydrolase Peptide
Residue Position
Phospholipase A2
1-123
CNBrl
1-8
Number of Residues
Amino acid sequence (partially or complete)
123
Ala-Leu-Trp-Gln-Phe-Asn-(Gly,Met)-
8
1 - - r - - T 1 1 7
.L
1
1
Ala-Leu-Trp-Gln-Phe-Asn-Gly-H.Ser 22----2---L
CNBr2
9-123
115
Ile-Lys-Cys-Lys-Ile-Pro-Ser-Ser-Glu-Pro-Leu2 3 - 2 2 2
16-21
6
22-43
22
Phe-Asn-Asn-Tyr-G1y-Cys-Tyr-Cys-G1y-Leu -
29-43
15
Cys-Gly-Leu-Gly-Gly-Ser-Gly-Thr-Pro-Val-Asp-Asp-Leu-Asp-Arg
41-43
3
'3
44-53
10
c7
53-58
6
Lys-Gln-Ala-Lys-Lys-Leu
T4
54-56
3
G1 n-A1 a-Lys
T5
57-62
6
Lys-Leu-Asp-Ser-Cys-Lys
60-81
22
'6
63-100
38
T6C2
70-73
4
T6C3
74-100
27
G4
82-87
6
G5
88-92
5
G6
93-114
22
'7
101-108
a
T8
109-116
8
Ser-Glu-Pro-Leu-Leu-Asp 2 2 - - - - - - - - . l 2
- A - A - J L L - - - ~ L - ~ L 2
Leu-Asp-Arg L 2 - 2 - - . l 2 2 - -
G3
Cys-Cys-Gln-Thr-His-Asp-Asn-Cys-Tyr-Lys - - - 2 - - A -
----2
----+L2----
Ser-Cys-Lys-Val-Leu-Val-Asp-Asn-Pro-Tyr- - - \ L 2 - 2 - - - - - \ A 2 - -
Val-Leu-Val-Asp-Asn-Pro-Tyr-( A
L
L
)-Asn-Asn-Tyr-Ser-Tyr-
3
Thr-Asn-Asn-Tyr ---22------\-
Ser-Tyr-Ser-Cys-Ser-Asn-Asn-Glu-Ile-Thr2 2 L - 3 -
I1 e-Thr-Cys-Ser-Ser-G1u 2----
Asn-Asn-A1a-Cys-G1u L
2
3
L
A
L
l
L
3
-22--\-1
Asn-A1a-A1 a-I1 e-Cys-Phe-Ser-Lys - 3 - 2 - 3
G7
L15-123
9
2
L
L
Ala-Phe-Ile-Cys-Asn-Cys-Asp-Arg-Asn-Ala-Ala-Ile-
Val -Pro-Tyr-Asn-Lys-Glu-(His ,Lys) - 2 - 2 - . A L 2 2 - . J
His-Lys-Asn-Leu-Asp-Lys-Lys-Asn-Cys
268
Bovine Phospholipase A, 5
15
10
20
25
a. Ala-Leu-Trp-Gln-Phe-Asn-Gly-Met-Ile-Lys-Cys-Lys-Ile-Pro-Ser-Ser-Glu-Pro-Leu-Leu-Asp-Phe-Asn-Asn-Tyr b. . . . . . Arg-Ser . . Ala Gly His . . Yet . . . . c. Val . . . Arg-Ser . . Gin Thr . . Asn . Lys Tyr . Glu . . Asp
. .
.
. .
35
30
.
40
.
55
. .
65
60
.
50
45
Gly-Cys-Tyr-Cys-Gly-Leu-Gly-Gly-Ser-Gly-Thr-Pro-Val-Asp-Asp-Leu-Asp-Arg-Cys-Cys-Gln-Thr-His-Asp-Asn Glu . . . . Glu. . . . . . . . . . . . . . . . . Glu Aia Val . .
. . . . . . . . . . . . . .
.
.
. . .
.
70
75
Cys-Tyr-Lys-Gln-Ala-Lys-Lys-Leu-Asp-Ser-Cys-Lys-Val-Leu-Val-Asp-Asn-Pro-Tyr-Thr-Asn-Asn-Tyr-Ser-Tyr Arg-Asp . . Asn . . . . . Phe Glu-Ser . . . Thr Glu Ser Arg-Phe Glu-Ser Lys-Phe
. .
. .
. . .
.
. .
. . . . . . . . . . . . . .
85
80
90
.
95
100
Ser-Cys-Ser-Asn-Asn-Glu-Ile-Thr-Cys-Ser-Ser-Glu-Asn-Asn-Ala-Cys-Glu-Ala-Phe-Ile-Cys-Asn-Cys-Asp-Arg . . . . Thr. . . . Asn. Lys. . . . . . . . . . . . . . . Gly-Thr Val . . . Asp-Lys . . . . . . . . . . . .
.
105
110
115
120
-
. . 125
Asn-Ala-Ala-Ile-Cys-Phe-Ser-Lys-Ual-Pro-Tyr-Asn-Lys-Glu-His-Lys-Asn-Leu-Asp - Lys Lys-Asn-Cys . . . . . . . . Ala. . . . . . . . . . Thr. . Tyr . . . . . . . . . Ala . . . Pro Asn . . . . Ser Arg Ala
.
.
.
.
Fig. 3 . Amino m i d sequence comparison of bovine, porcine and equine pancreatic phospholipase A , . Sequences are: ( a ) bovine; (b) porcine and (c) equine. Points indicate sequence homology with the bovine sequence. Deletions ( - ) have been assigned to the bovine and porcine enzyme
phospholipase A,, which differs from the bovine active center peptide by a Lys/Arg and a Gln/Glu substitution [7]. When both peptides were sequenced at the same time under identical conditions, cyclization occurred to a large extent at glutamic acid and not at glutamine. Therefore, we must conclude that in certain peptides it is not glutamine but glutamic acid which has the strongest tendency to cyclicize. The redigestion of peptide G, with staphylococcal protease was carried out with another batch of enzyme than the one used for the first incubation. In this second digest more non-specific cleavages occurred, some of which have a trypsin-like character. The cleavage of the Lys"-thialaminine" and Arg43thialaminine@ both occurred in a strongly basic part of the polypeptide chain. This non-specific behaviour has been described before [30], but we did not try to investigate whether this non-specificity is due to the presence of autoproteolytic products or to the presence of traces of contaminating enzymes. In Fig. 3 the primary structures of bovine, porcine and equine phospholipases are shown. The homology between the bovine and porcine enzymes and between bovine and equine enzymes is 84% and 73% respectively. About 719{ of the polypeptide chain is conserved in all three species. The homology between snake venom and pancreatic phospholipases has been shown in several papers [ 5 - lo]; for reasons of spacesaving these data are not repeated here. However, it should be noted that the number of residues present in the COOH-terminal portion of the enzyme, which is variable for the mammalian species, is fairly constant in the elapid enzymes, whereas in both classes of enzymes this region carries a large excess of positive
charges. This basic cluster may be more important for enzymatic activity than the length of this external [19] loop. Another remarkable feature is the asparagine at position 6 in the bovine phospholipase A,, where arginine is found in the porcine and equine enzymes and lysine is found in most elapid enzymes. Perhaps the absence of a positive charge can be the cause of the lower activity and penetrating power of bovine phospholipase A2 as compared to porcine and equine enzymes (see below), since the N-terminal is of importance for substrate binding [31,32]. Since the homology between the mammalian phospholipases is rather high, a solution of the X-ray structure of the bovine enzyme can also be applied successfully to phospholipases from other sources. We wish to thank Drs A. M. Municio (Madrid) and C. E. Dutilh for their help in preliminary investigations, Mr W. C. Puijk for technical assistance in carrying out the amino acid analyses and D r H. Wang for carefully reading the manuscript. D r R. Walter kindly provided us with a sample of post-proline cleaving enzyme and synthetic substrate. This study was carried out in part under the auspices of The Netherlands Foundation of Chemical Research (S.O.N.) and with financial aid from The Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
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269
E. A. M . Fleer. H. M . Verheij, and G. H. de Haas 4. Lowry, P. H.. Sarmiento, L. & Mitchell, H. K. (1971) Arch. Biochem. Biophys. 145. 338 - 343. 5. Halpert. J. & Eaker. D. (1975) J . Biol. Chem. 250, 6990-6997. 6. Tsao. F. H. C., Keim, P. S. & Heinrikson, R. L. (1975) Arch. Biochem. Biophys. 167, 706-717. 7. Puijk, W. C., Verheij. €I. M . & de Haas, G. 14. (1977) Bioc.hin1. Biopliys. Acta. 4Y2, 254- 259. 8. Evenberg, A,, Meijer, H.. Gaastra. W., Verheij, H. M . & de Haas, G. H. (1977) J . Biol. Chem. 252, 1189-1196. 9. Joubert. F. J. (1975) Eitr. J . Biochem. 52, 539 - 554. 10. Botes. D. P. and Viljoen, C. C. (1974) J . Biol. Chem. 249, 3827 3835. 11. Wells, M. A. (1971) Biochemistry. 10, 4074-4078. 12. Wells. M.A. (1973) Biochemisrry. 12, 1086-1093. 13. Slotboom. A. J., Pieterson, W. A,. Volwerk, J. J. & de Haas. G . H. (1976) Lipids (Paoletti, R., Bucellati, G. & Jacini. G.. eds) vol. I , pp. 99-107. Raven Press, New York. 14. Volwerk. J. J., Pieterson. W. A. & de Haas. G. H. (1974) Biochemisfry, 13, 1446- 1454. 15. Pieterson, W. A,, Vidal, J. C.. Volwerk, J. J . & de Haas. G. H. (1974) Biocheniistrv, 13, 1455- 1460. 16. de Haas, G. H.. Bonsen, P. P. M.. Pieterson, W. A . & van Deenen L. L. M. (1971) Biochim. Biophys. Acfa, 239, 252266 ~
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E. A. M. Fleer. H. M. Verheij, and G. H. de Haas. Biochemisch Laboratorium. Rijksuniversiteit te Utrecht Transitorium 3. Universiteitscentrum "Lie Uithol”, Padualaan 8, Utrecht. The Netherlands
