Detailed Biochemical Characterization of Human Placental Cystatin (HPC)

Placenta (2006), 27, 822e831 doi:10.1016/j.placenta.2005.09.005

Detailed Biochemical Characterization of Human Placental Cystatin (HPC) F. Rashid, S. Sharma and B. Bano* Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh, UP 202 002, India Paper accepted 10 September 2005

A low molecular weight thiol protease inhibitor (12,500) purified from human placenta has been characterized in detail. Human placental cystatin (HPC) was found to be stable in the pH range 3.0e9.0 and temperature stability was between 40 and 100 (C. It does not have any disulphide groups and carbohydrate content. There was no cross-reaction between anti-HPC serum and other purified cystatins like HMW kininogens isolated from sheep plasma and phytocystatins isolated from Phaseolus mungo. The kinetics of inhibition of HPC was studied with ficin and bromelain and the comparison was made with our already reported results with papain. The respective Ki values obtained for ficin and bromelain are 8.4 ! 10ÿ8 M and 9.5 ! 10ÿ8 M, respectively, whereas the value for papain was 5.5 ! 10ÿ8 M. The values of association constants (Kass) for ficin and bromelain were 2.9 ! 103 and 8.6 ! 102 Mÿ1 sÿ1, respectively, however, the value for papain was 3.4 ! 104 Mÿ1 sÿ1, the respective dissociation constant values for ficin and bromelain were 2.6 ! 10ÿ5 and 2.1 ! 10ÿ5 sÿ1, respectively, and the value obtained for papain was 2.3 ! 10ÿ5 sÿ1. These kinetic parameters taken together along with t1/2 values and IC50 values imply that HPC binds more effectively to papain, then ficin and least with bromelain. Far-UV-CD analysis shows that HPC has 21.08% a-helical structure and significant amount of b structure. Near-UV-CD spectra of HPC show positive peak at 280 nm indicating significant amount of tertiary interactions. The partial amino acid sequence analysis shows that HPC has highest sequence homology with chicken cystatin and Gly residue is present at position 11 rather than at conserved position 9, which has also been reported for human stefin A structure. The hydropathy plot of 1e30 amino acid residues shows that most amino acids of this stretch are present in the hydrophobic core of the protein. Owing to low molecular weight, absence of disulphide bonds and carbohydrate content HPC can be placed in type I cystatin family with some resemblance to chicken cystatin as shown by CD studies and amino acid sequence analysis. Placenta (2006), 27, 822e831 Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Human placental cystatin; Immunodiffusion; Kinetics of inhibition; Amino acid sequence; CD spectroscopy

INTRODUCTION Natural inhibitors of cysteine proteinases include the members of cystatin superfamily comprising of stefins, cystatins and kininogens [1,2]. Cystatins constitute a powerful regulatory system for endogenous cysteine proteinases, which may otherwise cause uncontrolled proteolysis and tissue damage. The name cystatin was given to superfamily of evolutionary, structurally and functionally related proteins involved in the inhibition of cysteine proteinases. All cystatins form a single superfamily within which all the members can be shown to have a statistically significant relation with each other or with papain but they do not resemble members of other superfamilies [3]. On the basis of sub-cellular localization, disulphide bonds, number of amino acid residues and sequence * Corresponding author. Tel.: C91 571 270 0857; fax: C91 571 270 2587. E-mail addresses: [email protected], [email protected] (B. Bano). 0143e4004/$esee front matter

homology, thiol proteinase inhibitors have been classified into three families [4,5]. Family 1 includes cystatins A, B and stefin C containing about 100 amino acid residues (11,000e12,000 Da) and lack disulphide bridges and carbohydrate content [6], they are mainly present in extra cellular fluids. Family 2 cystatins comprise of cystatins C, D, S, SN and SA and are found both in the cells and body fluids. They have about 115 amino acid residues, carbohydrate content and two disulphide loops towards the carboxyl terminal [7]. Family 3 cystatins represent more complex members which contain three family 2 like domains, they are the kinin precursors [8]. Their composite sequences are comprised of about 335 amino acid residues. They have additional disulphide bonds and the molecules are glycosylated. Cystatins form potent, non-covalent competitive inhibitors of cysteine proteinases of the papain superfamily [9,10]. The unique properties and wide range of physiological functions of thiol proteinase inhibitors are remarkable and demand attention. A proteinase inhibitor is of physiological importance Ó 2005 Elsevier Ltd. All rights reserved.

Rashid et al.: Human Placental Cystatin Studies

because inhibition is achieved at physiological concentration of the inhibitor in a sufficiently short time with negligible dissociation of the complex [11]. They have important regulatory role in normal body processes involving cysteine proteinase activity such as renal failure [12], osteoporosis [13], rheumatoid arthritis [14], septic shock [15], metastasizing cancer [16], purulent bronchiectasis [17], peridontitis [18] resulting due to imbalance between endogenous inhibitors and cysteine proteinases. A deficiency state in which levels of the intracellular cystatin are decreased due to mutation has recently been shown to cause progressive myoclonus epilepsy [19]. Cystatins have been purified from bovine muscle [20], human spleen [21], liver [22], blood polymorphonuclear granulocytes [23,24], amyloid fibrils [25] and saliva [26,27]. In continuation of the study of cystatins, the present communication describes detailed characterization of cystatin from human placenta, an organ of special importance in the human system. In normal pregnancy and in various pregnancy associated disorders, placenta plays highly significant role [28]. Lysosomes present in the cells of placenta take part in intracellular degradation of proteins [29], the activity of lysosomal enzymes (cathepsins B, H, L and S) is controlled by cystatins present in the placenta. Since lacuna exist in the isolation and physiochemical characterization of cystatin from human placenta, it was envisaged that a thorough and systematic study will be useful in understanding in depth about the placental cystatin and to compare its properties with other known cystatins. We have already reported interaction of this cystatin with papain in terms of spectroscopic methods and various kinetic parameters [30]. We have also reported inactivation and unfolding mechanism of HPC in presence of denaturants, GdnHCl and urea [31]. Here we report the detailed characterization of human placental cystatin given in terms of physicochemical properties, secondary and tertiary structure analysis by far- and near-UV-CD spectroscopy, kinetic parameters with various proteases, immunological properties and partial amino acid sequencing.

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homogenized in a buffer (400 ml) of neutral pH containing 1% NaCl, 3 mM EDTA, and 2% n-butanol. After centrifugation (3500 g for 10 min at 40 (C), the supernatant was adjusted to pH 3.0 by acetic acid and then incubated at 60 (C for 10 min to remove some unwanted proteins. The pH of the solution was brought to 6.0 with 3 M NaOH. After centrifugation (3500 g) for 15 min, the supernatant was fractionated between 50% and 80% ammonium sulphate saturation, the precipitated protein was dissolved in 20 mM sodium phosphate buffer, pH 6.0, and dialyzed against the same buffer containing 0.15 M NaCl. The dissolved protein precipitate was subjected to Sephadex G50e80 gel filtration chromatography. Molecular weight determination The molecular weight of the purified inhibitor was calculated by running marker proteins along with the inhibitor on SDSPAGE. The molecular weight under native conditions was determined by passing various marker proteins and the inhibitor on Sephadex G50e80 column. Assay of thiol proteinase inhibitory activity The inhibitory activity of cystatin was assessed by its ability to inhibit caseinolytic activity of papain by the method of Kunitz [32]. Inhibitory effects of cystatin on ficin, bromelain, trypsin and chymotrypsin were measured using casein as substrate. The protein concentration was determined by the method of Lowry et al. [33]. Thiol group estimation For determination of thiol groups of cystatin, the procedure given by Ellman [34] was used, using DTNB and molar extinction coefficient of 13,600 Mÿ1 cmÿ1. Carbohydrate estimation The carbohydrate content was analyzed by the method of Dubois et al. [35] using glucose as standard.

MATERIALS AND METHODS Materials Papain, ficin, bromelain, trypsin, chymotrypsin, Sephadex G50e80, casein, and acrylamide, bis-acrylamide, ethylene diamine tetra-acetic acid (EDTA), cysteine, anti-rabbit alkaline phosphatase (conjugate) and p-nitrophenyl phosphate were from Sigma chemical company, St. Louis, USA. Low molecular weight markers, Fruend’s complete and incomplete adjuvants were from GENEI India Limited. All other chemicals used were of highest purity grade available commercially. Methods Purification of human placental cystatin Purification of the cystatin was achieved by the method of Rashid et al. [30]. Fresh placental tissue (200 g) was

pH stability Fifty micrograms of the inhibitor was incubated with buffers of different pH values like 20 mM sodium phosphate buffer (pH 7.0e8.0), 20 mM sodium acetate buffer (pH 3.0e6.0) and triseHCl buffer (pH 9.0) for 30 min at 37 (C. Aliquots of this mixture were used for determination of remaining % inhibitory activity as described in the section of assay of proteinase inhibitory activity. Thermal stability (a) Fifty micrograms of inhibitor in 20 mM sodium phosphate buffer (pH 6.0) was incubated at various temperatures for 30 min. These samples were rapidly cooled in ice cold water bath and checked for residual activity against 50 mg of papain.

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(b) Fifty micrograms of the inhibitor at 90 (C was incubated for different time intervals, rapidly cooled and residual inhibitory activity measured against papain.

Immunological properties (a) Production of antiserum: Antibodies against human placental cystatin were raised by injecting 300 mg of purified inhibitor in Fruend’s complete adjuvant subcutaneously into healthy male albino rabbits. The injection was repeated every week in Fruend’s incomplete adjuvant and the rabbit was bled every second week. The blood collected was allowed to coagulate at 22 (C for 3 h. The antisera were decomplimented at 57 (C for 30 min and stored at ÿ20 (C in small aliquots. (b) Immunodiffusion: Immunodiffusion was performed essentially by the method of Ouchterlony [36]. One percent agarose in normal saline containing 0.2% sodium azide was poured on glass plate and allowed to solidify at room temperature. Fifteen microlitres of suitably diluted antiserum and required amount of antigen prepared in normal saline were added in different wells. The reaction was allowed to proceed for 12e24 h in a moist chamber at room temperature. The sheep HMW kininogen [37] and Phaseolus mungo (Urd e a commonly used Indian legume) cystatin [38] for immunodiffusion studies were purified in our laboratory. (c) Direct binding ELISA: The generation of antigen specific antibody was measured in the sera of HPC immunized rabbits by the technique of direct binding ELISA as given by Voller et al. [39]. Ninety-six wells of micro-titre plate (immulon 2HB, Dynex, USA) were coated overnight with 100 ml of antigen at 4 (C. The plate was washed three times with TBS-T buffer (Tris buffered saline Tween 20, pH 7.4, 20 mM Tris, 14.3 mM sodium chloride, 200 mg potassium chloride and 5 ml Tween 20 dissolved in 1 l of distilled water and pH adjusted to 7.4 by 1 N HCl). The unoccupied sites were saturated by incubation with 150 mg/200 ml of 1.5% milk in TBS (Tris buffered saline, pH 7.4, 20 mM Tris, 150 mM sodium chloride) for 5e6 h at room temperature. Plates were washed twice with TBS-T. The test and the control wells were then loaded with 100 ml of serially diluted serum. The plate was incubated for 2 h at room temperature and then overnight at 4 (C. One hundred microlitres of appropriate conjugate of anti-rabbit alkaline phosphatase (1:3000) is coated in each well and kept for 2 h at room temperature. After regular washing with TBS-T and distilled water, the substrate p-nitrophenyl phosphate (5 mg/100 ml of bicarbonate buffer, pH 9.5, 50 mM containing 0.02% sodium azide) was added in each well and incubated for 30e45 min. The reaction was stopped by addition of 100 ml of 3 M NaOH in each well. The absorbance of each well was monitored at 405 nm on a Qualigens ELISA reader.

Kinetics of inhibition The kinetics of inhibition for purified cystatin with papain has already been reported in our earlier communication [30]. The determination of inhibition constant (Ki), dissociation constant (Kdiss), association constant (Kass), IC50 values and half-life of enzymeeinhibitor complex (t1/2) with ficin and bromelain are given here. The data are compared with papain. (a) Determination of inhibition constants (Ki) of the human placental cystatin using ficin and bromelain: Ki determinations were carried out by lowering the enzyme concentrations to obtain a non-linearity of doseeresponse curves. Ficin and bromelain were used at a concentration of 0.05 mM to react with inhibitor in varying concentrations from 0.011 to 0.211 mM. The experiments were carried out at four different substrate concentrations [S] Z 0.5Km, 1Km, 2Km, 3Km and with Km Z 2.4 mM. The results were analyzed by the procedure of Krupka and Laidler [40] and Handerson [41]. (b) Determination of association rate constants (KC1): KC1 values were determined by measuring the time dependence of association under second-order conditions. Equimolar amounts of enzyme and inhibitor (0.05 mM) were mixed in a total volume of 1 ml of the same buffer of pH 7.0. Residual activity was assayed as a function of time after 0, 2, 5, 10, 15, 20, 25 and 30 min as described above. (c) Determination of dissociation rate constants (Kÿ1): Conditions for maximum association between the protease and the inhibitor were achieved before the reaction was shifted towards association by adding excess of substrate which binds the entire free enzyme. Substrate induced dissociation was monitored with identical enzymeeinhibitor complex incubated for 30 min at 37 (C. Excess substrate (6% casein) was added to the mixture for different time periods and then it was assayed for enzymatic activity. Kÿ1 and half-life of the complex are related by the equation t1/2 Z 0.693/Kÿ1.

CD spectroscopy Circular dichroism measurements were carried out at 25 (C on a Jasco Spectropolarimeter model J-720 using a SEKONICXY Plotter (model SPL-430 A). The concentration of inhibitor for far-UV-CD analysis was 23.2 mM and for near-UV-CD analysis the concentration was 80 mM. The path lengths used were 0.1 cm for far-UV-CD analysis and 1 cm for near-UVCD analysis. Amino acid analysis The sequencing of N-terminal 30 amino acid residues was done. Sequencing was performed on Shimadzu ppsq-21 Sequencer which employs Edman degradation to sequentially

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cleave and identify amino acids starting at amino terminus of the protein [42]. The highly purified protein was transferred to the PVDF membrane by Western blotting before analysis. Hydropathy plot The hydropathy profiles were calculated using the mean segment approach, i.e. determining the average hydropathy within a segment length of four residues, the segment being advanced one residue at a time from the sequence of N-terminal 30 residues [43]. RESULTS AND DISCUSSION Purification of the inhibitor As reported earlier [30] our scheme of purification of cystatin from human placenta which included ammonium sulphate precipitation and gel filtration chromatography on Sephadex G50e80 column is a rapid and simple procedure which gave a yield of 14.8% with 8913-fold purification. The precipitate obtained after 50e80% ammonium sulphate saturation was chromatographed on Sephadex G50e80 (Figure 1). Out of the two protein peaks in which peak one had very low thiol proteinase inhibitory activity, whereas second peak had significant inhibitory activity and protein content. The fractions corresponding to the peak II were pooled and lyophilized for further studies. The protein was found to be homogenous on the basis of charge and molecular weight [30].

Molecular weight The molecular weight was determined by SDS-PAGE as shown in Figure 2(A). The relative mobility (Rm) of each marker was plotted against log molecular weight (Figure 2(B). The molecular weight obtained was 12,445. It was further confirmed by passing marker proteins through Sephadex

G50e80 column. The Ve/Vo ratio was plotted against log M (Figure 3). From the overall data, the molecular weight was calculated to be 12,500. Cystatins type 1 and type 2 have been classified on the basis of molecular weight, presence or absence of disulphide bonds and carbohydrate content [6]. Cystatins from tissues are usually small inhibitors having molecular weights in the range of 11,000e25,000 [10]. Molecular weights of 11,400 and 12,000 have been reported for cysteine proteinases inhibitors isolated from human spleen [21], 14,000 and 14,300 for cystatins from bovine muscle [44], 14,200 from human liver [22]. For type 1 cystatins stefins A and B molecular weights of 11,100 and 12,000, respectively, have been reported [45]. Hence, the molecular weight of HPC obtained in present study suggests it to be type 1 cystatin. Hydrodynamic parameters for the HPC as determined from gel filtration behavior suggested a Stoke’s radius of 14.5 A˚ (Figure 4) as determined by the method of Andrews [46] and Laurent and Killander [47] and diffusion coefficient of 1.53 ! 10ÿ14 cm2/s. Diffusion coefficient depends on the size and shape of the molecule. The Stoke’s radius of cytochrome c is 16.4 A˚, it is a compact and globular protein. Thus, as indicated by the hydrodynamic properties human placental cystatin has a compact globular structure. Generally, cystatins type 1 and type 2 which are isolated from tissues lack carbohydrate content [48], whereas presence of carbohydrate content is a distinguishing property of type 3 cystatins, the kininogens [49]. However, presence of carbohydrate has been reported in some type 2 cystatins, for example, rat cystatin C isolated from urine is slightly glycosylated [50]. Ni et al. [51] found carbohydrate attachments in cystatin E. Glycosylation has also been demonstrated in cystatin F and cystatin M which are all type 2 cystatins [52,53]. Human placental cystatin is devoid of any carbohydrate content and disulphide bonds are also absent which is again a characteristic similar to type 1 cystatins [10].

0.35

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O.D at 660 (mn)

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Elution volume (ml) Figure 1. Gel filtration profile of the dialyzed and concentrated inhibitor extract on Sephadex G50e80. Eluted fractions were analyzed for their inhibitory activity against papain (–-–) and absorbance at 660 nm (– –) was taken.



Placenta (2006), Vol. 27

826 3.5

A 3 2.5

A Ve/Vo

A

B

HPC

2

B

C

1.5

D

E

1

C

0.5 0 0

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Log M Figure 3. Plot of log M vs Ve/Vo for markers passed through Sephadex G50e80 column: A, insulin (5.7 kDa); B, cytochrome c (12.7 kDa); C, ribonuclease (14 kDa); D, soyabean trypsin inhibitor (20.1 kDa); and E, papain (23 kDa). The arrow shows position of HPC.

E

a

reported similar stability for LMW-CPI up to 100 (C for 5 min.

b

Immunological properties

B A 1.6

B 1.4

Log M

C 1.2

D HPC

1.0 E

0.8

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The placental cystatin showed moderate immune response and gave a low titre of antibodies in rabbits owing to its low molecular weight. The titre of antibodies determined by direct binding ELISA in rabbit serum was 2238.73 (Figure 8). The low antibody titre suggests that the inhibitor used as antigen here is of low molecular weight range. The antibodies raised against purified cystatin gave a reaction of identity with the inhibitor as indicated by a single precipitin line on immunodiffusion indicating that HPC has immunogenic purity and homogeneity (Figure 9). The antisera exhibited no immunogenic identity with sheep plasma HMW kininogens and phytocystatin purified from Phaseolus mungo (Urd e a commonly used Indian legume) (Figure 10). This indicates that the

Figure 2. (A) SDS-PAGE of cystatin with protein molecular weight markers: A, ovalbumin (43 kDa); B, carbonic anhydrase (29 kDa); C, soyabean trypsin inhibitor (20.1 kDa); D, lysozyme (14.3 kDa); and E, aprotinin (6.9 kDa). Lane I e markers, lane II e purified HPC. (B) Plot of log M vs relative mobility (Rm) of markers for determination of molecular weight of cystatin. The arrow indicates position of HPC.

10.0 9.0 8.0

Stability to pH and temperature Effect of pH on human placental cystatin activity was examined at various pH values. Figure 5 shows that the inhibitor is stable in the pH range 3.0e9.0. Further, this inhibitor was found to be stable in the temperature range 40e100 (C (Figure 6). The inhibitor was stable for 120 min at 90 (C (Figure 7). These properties are in accordance with other cystatins, cystatin from bovine muscle [20] and chicken cystatin [54]. Zerovnik et al. [55] have shown high thermal stability of chicken cystatin and stefins A and B. Barrett et al. [56]

-(Log Kav)1/2

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Stoke's radius (r) Figure 4. Plot of ÿ(log Kav)1/2 vs r e Stoke’s radius for the following markers: A, cytochrome c (16.4 A˚); B, ribonuclease (19.2 A˚); and C, soyabean trypsin inhibitor (22.6 A˚). The arrow indicates position of HPC.

Rashid et al.: Human Placental Cystatin Studies

827 120

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40 60 20 50 2

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pH Figure 5. pH Stability profile of the HPC. Fifty micrograms of the inhibitor was incubated in 20 mM sodium acetate buffer, pH 3.0e6.0, sodium phosphate buffer, pH 7.0e8.0, triseHCl buffer, pH 9.0, for 30 min at 37 (C. Remaining % inhibitory activity was analyzed against 50 mg papain at 37 (C.

human placental cystatin has no immunogenic identity or similarity in structure and composition to high molecular weight mammalian kininogens (type 3 cystatins) and to phytocystatins. Kinetics of inhibition The human placental cystatin (HPC) strongly inhibits thiol proteinases papain, ficin and bromelain but does not inhibit serine proteinases like trypsin and chymotrypsin. This has also been reported for many other cystatins along with the thiol proteinase inhibitor isoforms purified from human spleen [21]. The inhibitory activity of HPC towards above mentioned thiol proteinases and serine proteinases was examined using casein as substrate. Accurate Ki values were determined by working at lower enzyme concentrations and using equations derived by Voller et al. [39], Krupka and Laidler [40] and

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Time (min) Figure 7. Thermal denaturation of the inhibitor at 90 (C. Fifty micrograms of HPC in 20 mM sodium phosphate buffer, pH 7.0, was incubated for different time intervals, rapidly cooled and % inhibitory activity measured against 50 mg papain.

Handerson [41]. Ki values were calculated from the slope of inhibition of caseinolytic activity of papain, ficin and bromelain (Table 1). HPC is a potent inhibitor of thiol proteinases as is evident by their Ki values. It was readily apparent that the degree of inhibition varied with absolute concentration of the inhibitor as is expected for tight binding reversible inhibition. The data show that HPC inhibited papain, ficin and bromelain with Ki values of 5.5 ! 10ÿ8 M, 8.4 ! 10ÿ8 M and 9.5 ! 10ÿ8 M, respectively, under conditions of routine

60

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Temperature (ºC) Figure 6. Effect of temperature on HPC. Fifty micrograms of inhibitor in 20 mM sodium phosphate buffer, pH 7.0, was incubated at various temperatures for 30 min. Remaining % inhibitory activity was analyzed against 50 mg papain.

Figure 8. Direct binding ELISA: serially diluted antiserum and pre-immune serum were incubated with 0.5 mg/100 ml antigen. The procedure has been described in Methods. The curve with solid circles is for post-immunized sera, whereas the curve with hollow circles is for pre-immunized sera.

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Table 1. Kinetic constants obtained on interaction of human placental cystatin with proteinases e papain, ficin and bromelain Kinetic parameters Ki (M) KC1 (Mÿ1 sÿ1) Kÿ1 (sÿ1) IC50 value (mM) Half-life of the complex

Figure 9. Ouchterlony immunodiffusion e anti-human placental cystatin antiserum was raised in rabbits. For the immunodiffusion study, the antiserum is allowed to react with the inhibitor (60 mg) on agarose plates as described in the Methods section. The central well contained the antiserum, whereas the surrounding three wells contained human placental cystatin.

assay system. The Ki value is lowest for papain, hence it has the highest affinity for the inhibitor. These values are in good comparison with other thiol proteinase inhibitors, for example the inhibition constant (Ki) for chicken cystatin and cystatin C has been reported to be 5 ! 10ÿ12 M with papain, Ki values of nanomolar range have been reported for cathepsins B, H and L with cystatin A [48], chicken cystatin [49], cystatin C [57] and cystatin D [58]. The increasing value of Ki with an increase in substrate concentration suggests the inhibition to be competitive. This finding is supported by the apparent results of Nicklin and Barrett [59] for the inhibition of human cathepsin B by chicken cystatin. They obtained Ki (approximately) values of 1.85 and 3.68 nM with the substrate concentration of 0.05 and 0.39 mM, respectively. IC50 value is the concentration of the inhibitor at which 50% of the enzyme is inhibited. IC50 values of the human placental cystatin for the three proteinases papain, ficin and bromelain were 0.051, 0.091 and 0.131 mM, respectively.

Papain

Ficin

Bromelain

5.5a G 0.02 ! 10ÿ8 3.4 G 0.01 ! 103

8.4 G 0.1 ! 10ÿ8 2.9 G 0.3 ! 103

9.5 G 0.2 ! 10ÿ8 8.6 G 0.1 ! 102

2.3 G 0.02 ! 10ÿ5 0.051

2.6 G 0.3 ! 10ÿ5 0.091

2.1 G 0.1 ! 10ÿ5 0.171

3.01 ! 104

2.6 ! 104

3.3 ! 104

a Results represent the mean G SEM calculated from three independent experiments.

Lesser IC50 value suggests a greater affinity for the inhibitor towards the enzyme. Thus, these values indicate that the affinity of HPC for these proteinases are in the order of papain O ficin O bromelain. Katunuma and Kominami [60] have found the IC50 value of 0.16 mg of the inhibitor isolated from rat liver for papain and 0.46 mg for ficin and IC50 value of 4.2 mg for cathepsin B and 0.14 mg for cathepsin H. The kinetics of association was determined assuming that the enzymes and the inhibitor react in such a way that dissociation rate is low enough to neglect the reverse reaction during the initial part of the reaction. The slope of the straight line obtained by plotting 1/[E] vs time for papain, ficin and bromelain gave KC1 values of 3.4 ! 104, 2.9 ! 103 and 8.6 ! 102 Mÿ1 sÿ1, respectively. Again the affinity was in the order of papain O ficin O bromelain. Bjork and Ylinenjarvi [61] have reported association constant value for chicken cystatin which is 9.9 ! 106 Mÿ1 sÿ1 with papain. Earlier, Nicklin and Barrett [59] gave KC1 values of 1 ! 107 and 6.3 ! 106 Mÿ1 sÿ1 for chicken cystatin with papain and dipeptidyl peptidase I, respectively, and 4.3 ! 106 Mÿ1 sÿ1 with ficin. The dissociation constant values (Kÿ1) for the enzyme einhibitor complex were determined by displacement procedure, in which the inhibitor released from the complex was 40 30

CD (mdeg)

20 10 0 -10 -20

Figure 10. Immunodiffusion of human placental cystatin, sheep HMW kininogens and Phaseolus mungo cystatin. The central well contains the antiserum, whereas the wells 1, 2, 3 and 4 contain human placental cystatin, sheep HMW kininogen, Phaseolus mungo (urd) cystatin and saline, respectively.

-30 200

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Wavelength (nm) Figure 11. Far-UV-CD spectra of native HPC. The concentration of inhibitor for far-UV-CD analysis was 23.2 mM and the path length was 0.1 cm.

Rashid et al.: Human Placental Cystatin Studies

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20

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trapped by excess substrate (casein) with the increase in time. The amount of enzyme released from the complex was monitored by continuous measurement of enzyme activity. The Kÿ1 values obtained for papain, ficin and bromelain were 2.3 ! 10ÿ5, 2.6 ! 10ÿ5 and 2.1 ! 10ÿ5 sÿ1, respectively. The values of Kÿ1 are consistent with Kÿ1 values obtained for chicken cystatin, 5 ! 10ÿ5 sÿ1 with papain and ÿ3 ÿ1 2.3 ! 10 s with cathepsin B [59]. The calculated half-life values of enzymeeinhibitor complex using these Kÿ1 values for papain, ficin and bromelain were 3.01 ! 104, 2.6 ! 104 and 3.3 ! 104 sÿ1, respectively. The above data give comprehensive information about the kinetics of inhibition of purified cystatin with ficin and bromelain, and the comparison with the values obtained earlier with papain [30] overall shows that this inhibitor inhibits papain more effectively than other two proteinases. CD spectroscopy CD spectra in the far-UV region depict the contribution of the secondary structure of the protein [62]. The a-helical

4-8

8-12

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Residue Number Figure 13. Hydropathy plot of the N-terminal 30 amino acid stretch of HPC. The average hydropathy index of four residues was plotted starting from residues 1e4, 4e8, 8e12, 12e16, 16e20, 20e24, 24e28.

structure of the protein in the far-UV region is characterized by negative peaks at 208e210 nm and 222 nm and a positive peak between 190 nm and 192 nm [62,63]. In the present study we measured far-UV-CD spectra at a concentration of 23.2 mM to get a better and well defined spectrum in the far-UV region (Figure 11). At this standardized concentration, the spectra show a peak at 222 nm and a small peak at 208 nm also. HPC has a-helical content of 21.08% and also shows presence of significant amount of b structure. The a-helical content was calculated from the ellipticity values at 222 nm using equation given by Chen et al. [63]. This type of structure has also been reported for chicken cystatin which has a-helical content of about 20% and substantial amount of b structure [64]. Structurally, the resemblance between chicken cystatin which is a type 2 cystatin and stefins A and B which are type 1 cystatins has been reported earlier [65,66]. Stefin A and stefin B have an a-helical content of 22% and 20%, respectively, however, chicken cystatin has disulphide bonds which are absent in HPC as well as in stefins A and B, in

Table 2. N-terminal amino acid sequence of HPC Human placental cystatin Y E D D E P V (HPC) Chicken cystatin (27.2%) Human salivary cystatin-SA W S (20.8%) Bovine cystatin (29%) Human cystatin C (20.8%) S Human salivary cystatin-S7 S S (16.6%) Human stefin A (16.6%) M Human stefin B (8.3%) M Rat liver TPI (8.3%) M Rat cystatin-SM (11.1%)

L L E G L I

R L D D G F V I

L G L I

W F G V/L Q

P

S E D R S R L L G A P V P V D E N D E G L K E E D R I I V G G I T N A D L N D E W V

Q Q

S S

R L P G G L M E A D V N E E G V P G K P P R L K G G P M D A S V E E E G V K E E N R I I Q G G I Y D A D L N D E W V

Q R Q

I P G G L S E M C G A P S A M C G A P S A G H

A T T F

M Q M L

P P P G

A A A G

T T T I

P A T E

E E E K

I T T S

Q Q Q S

E H E M

I I I E

V A A E

D D D E

K Q K G

V V V A

K R K S

The highly purified cystatin was transferred to PVDF membrane by Western blotting before doing amino acid analysis. Percent homology with other known cystatin N-terminal sequences has been given in parenthesis. The alphabets in boldface indicate those residues which are common in above given cystatins.

830

addition there are differences in amino acid sequence [67]. The near-UV-CD spectra of human placental cystatin are characterized by a positive peak around 280 nm (Figure 12) showing significant amount of tertiary structure in this inhibitor. The absence of positive peaks in the region 250 nm indicates that disulphide bonds are absent in the protein [68] which supports our earlier mentioned observation of inability to detect any free thiol groups in this inhibitor. Amino acid analysis The N-terminal 30 amino acid residues were sequenced. The sequence gave some interesting results. As reported for other human cystatins, the human placental cystatin also has Gly residue at position 11 rather than at the conserved position 9 in various species [69], however, maximum sequence homology was observed with chicken cystatin [64]. Sequence homology of HPC was also observed with bovine cystatin [70], human salivary cystatin SN, cystatin-S7, cystatin C [71,72,45], rat cystatin-SM [73], human stefin A [24] and stefin B [74], and rat liver TPI (thiol proteinase inhibitor)

Placenta (2006), Vol. 27

[75], the details are given in Table 2. Using the sequence of these N-terminal amino acid residues a hydropathy plot was made using the respective hydropathy indices of each individual amino acid (Figure 13). It was observed that among the 30 residues sequenced the stretch of 4e8, 8e12 and 20e24 residues has maximum average hydropathy index indicating that these residues are present inside the hydrophobic core of the protein. Thus, owing to absence of any carbohydrate content and disulphide bonds and all other results taken together, HPC can be placed in type 1 cystatin family with resemblance to chicken cystatin like other members of this family and with no structural similarity observed with type 3 cystatins/kininogens. Cystatins have important roles to play in normal body processes owing to their cysteine proteinase inhibitory activity. Our results have shed some light on the structure and function of this inhibitor, which certainly will add to the scientific information available about these inhibitors and help in understanding cystatins on the physicochemical basis.

ACKNOWLEDGEMENTS Facilities provided by Aligarh Muslim University are gratefully acknowledged. F.R. is a Senior Research Fellow of the Council of Scientific and Industrial Research, New Delhi, India. We are grateful to SAP-DRS and UGC-FIST programmes for their generous research support.

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