Bioscience Reports, Vol. 8, No. 2, 1988

Proteolytic Specificity of Chicken Cathepsin L on Bovine/3-Casein

Eric D u f o u r 1'3 and B r u n o R i b a d e a u - D u m a s z

Received February 29, 1988

The proteolytic specificity of chicken cathepsin L was studied using bovine fl-casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P~. Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P~-P~ positions and that the S~ subsite can interact with modified amino acids such as phosphoserine.

KEY WORDS: cathepsin L; specificity, fl-casein; proteolysis

ABBREVIATIONS: RP-HPLC, reverse phase high performance liquid chromatography; NMec, N-methyl cournarylamide; TEA, triethylamine; TFA, trifluoroacetic acid.

I N T R O D U C T I O N

The cysteine proteinase family includes mainly the plant thiol proteases, papain and actinidin, and animal lysosomal thiol proteases, cathepsins B, H and L to which amino acid sequences are closely related (1, 2). The characteristics of the papain active site have been investigated by Schechter and Berger (3) who have shown that the enzyme could bind a peptide along a length of seven residues. Each subsite of the active site accommodates one amino acid. The investigations on cathepsin L specificity are rather rare and, hitherto, there is no specific synthetic substrate for this proteinase. Cathepsin L specificity has only been investigated using peptides such as synthetic substrates (4, 5), insulin B chain (6, 7) and glucagon (8) which exhibit a limited range of peptide bonds.

In the present paper, cathepsin L specificity was studied using as a substrate bovine fl-casein (209 residues) which displays a wide diversity of peptide bonds, most of which seem to be accessible in the native protein. Ten peptides released during the digestion were purified and identified by amino acid composition and

1S.R.V., I.N.R.A. de Theix, 63122 Ceyrat, France and 2I.N.R.A. Jouy en Josas, 78350 Jouy en Josas, France.

3 To whom correspondence should be addressed.

185 0144-8463/88/0400-0185506.00/0 �9 1988 Plenum Publishing Corporation

186 Dufour and Ribadeau-Dumas

sequence analysis. Our study gives new information on the specificity of S~ and S~ which could be used to synthesize new substrates.

MATERIALS AND METHODS

Purification of Chicken Liver Cathepsin L

The avian enzyme was purified as described previously (2). Assay for cathepsin L was carried out according to Barrett (9) and one enzyme unit (U) was defined as the amount of proteinase hydrolysing 1 nmol NMec. m1-1 . rain -a.

Reaction with Bovine t-Casein

Bovine/3-casein was prepared as described by Mercier et al. (10). 2 ml/3-casein (5 mg. m1-1) in 0.05 M NH4CH3CO z buffer, pH 5.8, containing 2 mM DTT was incubated with 10 #1 cathepsin L (2 U . ml -~) at 37~ Aliquots (200 #1) were taken after 5, 20, 30, 60, 90 and 180 min, and the reaction was stopped by adding 20 #1 of 1 M iodoacetate. The residual/3-casein was precipited by lowering pH to 2.5 with TFA (1.1% final concentration) and the supernatant was recovered after centrifugation.

Fractionation of Peptide Mixtures

The 1.1% TFA-soluble fractions were analysed on a reverse phase C-18 column (/~Bondapak, 4.6mm ID • SFCC, Gagny, France) using a Waters HPLC system. Most fractionations were carried out with system I:(A) 0.115% TFA; (B) 0.1% TFA in 60% CH3CN. Some purifications were achieved with system II:(A) 0.03% TFA and 0.03% TEA; (B) 0.025% TFA and 0.025% TEA in 60% CH3CN. All separations were carried out at 40~ with flow rate of 2ml . min -1. The absorbance was recorded at 214nm. The manually collected fractions were dried under vacuum.

Peptide Identification

The amino acid composition of the peptides was determined after acid hydrolysis under vacuum in the presence of 5.7NHC1 for 24h at ll0~ and hydrolysates were run through an amino acid analyser (Biotronik LC 5000, Munich, FRG). Amino acid analyses were often sufficient to assign the proper location of peptides in the known sequence of bovine t-casein (11). The manual "partitioning method for small peptides" described by Tarr (12) was used for sequence determinations. The PTH amino acids were identified as reported elsewhere (2). In all cases the N-terminal three residues of peptides were sequenced.

Specificity of Cathepsin L 187

RESULTS AND DISCUSSION

Bovine fl-casein was incubated with cathepsin L. Aliquots were taken at intervals and the 1.1% TFA-soluble fractions were analysed by RP-HPLC. As shown in Fig. 1, peptide mixtures obtained at 5, 20, 30, 60, 90 and 180min incubations were separated on a C-18 column and the peptides were eluted by a linear gradient using system I. Due to its loose tertiary structure, fl-casein peptide bonds are readily accessible to proteases and fl-casein is rapidly hydrolysed by cathepsin L. The major peptides detected were numbered according to their time of appearance during the digestion (Fig. 1). Peaks 1 to 3 appeared after 10 min hydrolysis (not shown). Peak 1.2 was a mixture of two peptides (1 and 2) which were resolved with system II (Fig. 2). Nevertheless, with respect to peak heights and peptide lengths, peptide 2 might be released slower than peptide 1. After

J i

v ~

i

o

~ I . o

F ,

i i

( s / / "/~ / / ~1 ~I.A / , '", "/" Jl "//"~ 4 ~ ~

,,, . .

" i , , ....- .2

i, !'. Ill ?-.,'f

Fig. 1, Separation by RP-HPLC of the peptides from the 1.1% TFA-soluble fractions obtained after 5 rain (A), 20 min (B), 30 min (C), 60 min (D), 90 min (E) and 180 min (F) of hydrolysis of fl-casein by chicken cathepsin L. Elution was in system I as described in Materials and Methods. The peptides 1 to 10 were analysed (see Table 1 and Fig. 3).

188 Dufour and Ribadeau-Dumas

1.0 ~

T

0.5.

O.

2

• S• •••S•

100

,'7"

, 5 0

, 0

Tim. (~i~)

Fig. 2. Separation by RP-HPLC of the peptides 1 and 2 from the peak 1.2 obtained after 30 min hydrolysis using system II as described in Materials and Methods.

20 min incubation (Fig. 1B) peaks 4 and 5 appeared and after 60 min hydrolysis (Fig. 1D) the chromatograms exhibited peaks 6 to 8. Two well resolved peaks (9 and 10) appeared only at 3 h incubation (Fig. 1F). The purified fractions 1 to 10 have been identified by amino acid analyses and by Edman degradation (Table 1). Among these ten peptides, the stable cleavage products consisted of peptides 1, 2, 5, 8, 9 and 10 which were seen after extensive incubation with cathepsin L, whereas peptides 3, 4, 6 and 7 were further digested.

Peptide 3, an hexapeptide, was rapidly hydrolysed and disappeared totally after 60rain hydrolysis (Fig. 1D) suggesting that cathepsin L can accept a NH2-ffee amino acid in position P3. Unfortunately the products of hydrolyse of peptide 3 were not identified. However/3-casein contains in its C-terminal part an amino acid sequence encompassing residues 177-191 that is susceptible to proteolysis by cathepsin L and releases peptides 7 to 9: peptide 8 (Ala 189-Leu 191), appearing with peptide 7, and peptide 9 (Ala 177-Pro 179) are tripeptides. They are generated by the hydrolysis at the N-terminal part of larger peptides, e.g. after 3 h incubation peptide 7 is digested and releases the tripeptide 9 which agrees with a NH2-free amino acid in position P3 (Fig. 3).

The first bonds cleaved were Gin 34-P.Ser 35, Ala 53-Gin 54, Val 59-Tyr 60

Specificity of Cathepsin L 189

Table 1. Amino acid composition and determination of the N-terminal sequence of the peptides corresponding to peaks 1 to 10

Peptide N o . 1 2 3 4 5 6 7 8 9 10

A s x - - 2 . 0 ( 2 ) - - 1 .7 (2 ) - - - - 1 .0 (1) - - - - - - T h r - - 1 .0 (1) 0 . 9 ( 1 ) 1 .0 (1) - - 1 .0 (1) . . . . S e r - - 1 .0 (1 ) 1 .0 (1) 1 .0 (1) - - 0 . 8 (1 ) . . . . G l x - - 7 . 7 ( 8 ) 1 .9 (2) 7 .5 (8 ) - - 2 . 2 (2 ) 2 . 1 ( 2 ) - - - - 1 .1 (1) P r o 3 . 5 ( 4 ) 2 . 1 ( 2 ) -- 1 .5 (1 ) - - 4 . 6 ( 4 ) 3 .5 (3 ) - - 1 .0 (1) 2 . 3 ( 2 ) G l y 1 .0 (1) - - - - - - 1 .0 (1) . . . . . A l a - - 0 . 9 ( 1 ) - - 1 .0 (1) - - - - 1 .0 (1) 1 .0 (1 ) 1 .0 (1 ) - - V a l - - - - 1 .1(1) - - 1 .1 (1) 2 . 3 ( 2 ) 1 .3 (1) - - 1 .2 (1) 1 .2 (1) Met . . . . . 0 . 3 ( 1 ) 0 . 3 ( 1 ) - - -- --

I l e 0 . 8 ( 1 ) 0 . 9 ( 1 ) - - 0 . 7 ( 1 ) 1 .2 (2) - - 1 .0 (1) - - - - - - L e u -- 1 .2 (1 ) 1 .0 (1) 1 .1 (1) -- 2 .4 (2 ) -- 1 .2(1) - - --

T y r 1 .1 (1 ) . . . . . 1 .1 (1 ) - - - - 0 . 9 ( 1 ) Vhe 1 .1 (1 ) 0 . 9 ( 1 ) - - 1 .8 (2 ) 1 .1 (1) 1 .0 (1 ) - - 1 .0 (1) - - 1 .1 (1) His 0 . 9 ( 1 ) 0 .8 (1 ) - - 0 . 9 ( 1 ) . . . . . . L y s - - 1 .1 (1 ) - - 2 . 2 ( 2 ) . . . . . . A r g . . . . . . 1 .1 (1 ) -- -- --

N - t e r m i n a l sequence Y - P - F S - E - E - Q Q - T - Q K - F - Q G - P - F L - P - P A - V - P - Y A - F - L A - V - P Y - P - V

Peptide identified 6 0 - 6 7 3 5 - 5 3 5 4 - 5 9 3 2 - 5 3 2 0 3 - 2 0 9 1 5 1 - 1 6 3 1 7 7 - 1 8 8 1 8 9 - 1 9 1 1 7 7 - 1 7 9 1 1 4 - 1 2 0

and His 67-Asn 68 and released peptides 1, 2 and 3 which were linked together (Fig. 3). Peptide 4 (Lys 32-A1a 53) overlapped peptide 2 and differed by its N-terminal part. These results suggest that the most proteolytically sensitive regions in fl-casein are encompassing residues 35-67. Peptides 5, 6 and 7 were located in the C-terminal amino acids of fl-casein. It is a sensitive region for various proteases such as rennin (13) and cell wall proteinase from lactic acid bacteria (14) especially the Leu 192-Tyr 193 bond. Peak 10 was identified as peptide 114-120 (Figure 3).

I0 20 30 40 50

RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIH

L 2.

4. _

6O 70 80 90 i00

~ SL~YPFPGPI~NSLPQNIPPLTQTPWVPPFLQPEVMGVSKVKE 3 ,T i~..~

110 120 130 140 150

AMAPKHKEMPFP~YPVEPF~ESQSLTLTDVENLHLPLPLLQSWMHQPHQp ~..10---~

160 170 180 190 200

~ PPTVMFPPQSV~SLSQSKVLPVPQK~VPYPQRDMPI~AF~LYQEPVLGP 6 .... t ~, 7- f8J Lg~

209 VRGPFPIIV

t----5---~

Fig . 3. S i t e s o f h y d r o l y s i s by chicken cathepsin L and location of peptides 1 to 10 in fl-casein sequence. S*: phosphoserine.

190 Dufour and Ribadeau-Dumas

Table 2. Amino-acid residues around the scissile bonds

Peak No. Hydrolysed bonds P4 P3 P2 P1 P ' I P'2 P'3

1 Val 59-Tyr 60 Gin Ser Leu Val Tyr Pro Phe His 67-Asn 68 Gly Pro Ile His Asn Ser Leu

2 Gin 34-Ser 35 Glu Lys Phe Gin P. Ser Glu Glu 3 Ala 53-Gln 54 His Pro Phe Ala Gin Thr Gin 4 Glu 31-Lys 32 Lys Lys Ile Glu Lys Phe Gin 5 Arg 202-Gly 203 Gly Pro Val Arg Gly Pro Phe 6 Pro 150-Leu 151 Pro His Gin Pro Leu Pro Pro

Leu 163-Ser 164 Gln Ser Val Leu Ser Leu Ser 7 Lys 176-Ala 177 Val Pro Gin Lys Ala Val Pro 8 Gin 188-Ala 189 Met Pro Ile Gin Ala Phe Leu

Leu 191-Leu 192 - - Ala Phe Leu Leu Tyr Gin 9 Pro 179-Tyr 180 - - Ala Val Pro Tyr Pro Gln

10 Lys ll3-Tyr 114 Pro Phe Pro Lys Tyr Pro Val Thr 120-Glu 121 Glu Pro Phe Thr Glu Ser Gin

Table 2 reports the amino acids located at positions P1, P2, P3, P4 and P~, P~, P~, according to the nomenclature of Schechter and Berger (3). For cathepsin L, as for the other eysteine proteinases (9, 15), the amino acid residues in position Pz are essentially non-polar such as phenylalanine, isoleucine, leucine and valine, but we find proline and glutamine too (Table 2). Proline residues, which have been considered to be excluded from $1 and $2 subsites (16), are found in all positions except P~, but mainly in P3 and P~ positions; in position P~ we very often find gluatimine. In subsite S~ a broad diversity of amino-acid side chains appears to be accepted since we find, for peptides 1 to 5, Ala, Arg, Gln, Glu, His, Lys, Pro, and Val in this position. For the scissile bond Gln 34-P.Ser 35, P[ was a phosphoserine residue.

Among the amino acids around the cleaved bonds, Tyr-Pro pairs (60-61, 114-115 and 180-181) were found three times at positions P~-P~ (Table 2). It is an interesting point since there are only four tyrosine residues in the /3-casein sequence. The S~ and S~ subsites of cathepsin L may interact preferentially with a Tyr-Pro pair since the peptide 10 exhibits Tyr-Pro at positions P~-P~ and an atypical amino acid, a proline residue, in position P2. In the insulin B chain, the three bonds preferentially cleaved also involve a tyrosine at or near the site of cleavage (17), suggesting that this is a key feature.

The typical substrates used to assay cathepsins, e.g. peptidyl-(4-methyl)-7- coumarylamide are restricted in their selectivity because S~, S~ and S~ subsites are not taken into account. Our study on the hydrolyse of/3-casein by chicken liver cathepsin L gives new information on the specificity of subsites of the enzyme active site, and suggests that it would be very interesting to investigate the positions P~, P~ and P~ using new synthetic substrates.

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Specificity of Cathepsin L 191

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10. Mercier, J. C., Maubois, J. L., Poznanski, S. and Ribadeau-Dumas, B. (1968). Bull. Soc. Chim. Biol. 50: 521-530.

11. Ribadeau-Dumas, B., Brignon, G., Grosclaude, F. and Mercier, J. C. (1972). Eur. J. Biochem. 25: 505 -514.

12. Tarr, G. E. (1982). In: Methods in Protein Sequence Analysis (Elzinga, M., ed.) Humana Press, Clifton, NJ, pp. 223-232.

13. Caries, C. and Ribadeau-Dumas, B. (1984). Biochemistry 23:6839-6843. 14. Monnet, V., Le Bars, D., and Gripon, J. C. (1986). FEMS Microb. Lett. 36:127-131. 15. Schechter, I. and Berger, A. (1968). Biochem. Biophys. Res. Com. 32:898-902. 16. Kirschke, H. and Shaw, E. (1981). Biochem. Biophys. Res. Com. 44:454-458. 17. Gal, S. and Gottesman, M. M. (1986). Biochem. Biophys. Res. Com. 139:156-162.