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Originally published In Press as doi:10.1074/jbc.M000431200 on May 31, 2000

J. Biol. Chem., Vol. 275, Issue 33, 25547-25555, August 18, 2000
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Pro-sterol Carrier Protein-2

ROLE OF THE N-TERMINAL PRESEQUENCE IN STRUCTURE, FUNCTION, AND PEROXISOMAL TARGETING*

Friedhelm SchroederDagger §, Andrey FrolovDagger , Olga Starodub, Barbara B. AtshavesDagger , William Russell||, Anca PetrescuDagger , Huan HuangDagger , Adalberto M. GallegosDagger , Avery McIntoshDagger , Dana TahotnaDagger , David H. Russell||, Jeffrey T. Billheimer**, Charles L. BaumDagger Dagger , and Ann B. Kier

From the Dagger  Department of Physiology and Pharmacology,  Department of Pathobiology, and || Department of Chemistry, Texas A & M University, College Station, Texas 77843-4466, the ** Cardiovascular Department, DuPont Merck Pharmaceutical Company, Experimental Station 400-3231, Wilmington, Delaware 19898-0400, and the Dagger Dagger  Department of Medicine, Clinical Nutrition Research Unit and Section of Gastroenterology, University of Chicago, Chicago, Illinois 60637

Received for publication, January 18, 2000, and in revised form, May 25, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less alpha -helix, 7-fold more beta -structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (nanomolar Kd values) in the order: cholesterol straight chain fatty acid > kinked chain fatty acid. Laser scanning confocal microscopy and double immunofluorescence demonstrated that pro-SCP-2 was more efficiently targeted to peroxisomes. Transfection of L-cells or McAR7777 hepatoma cells with cDNA encoding pro-SCP-2 resulted in 45% and 59% of SCP-2, respectively, colocalizing with the peroxisomal marker PMP70. In contrast, L-cells transfected with cDNA encoding SCP-2 exhibited 3-fold lower colocalization of SCP-2 with PMP70. In summary, the data suggest for the first time that the 20-amino acid presequence of pro-SCP-2 alters SCP-2 structure to facilitate peroxisomal targeting mediated by the C-terminal SKL peroxisomal targeting sequence.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite its discovery nearly 2 decades ago (1), the physiological function of sterol carrier protein-2 (SCP-2)1 remains an enigma. Although experiments performed in vitro as well as studies with transfected cells strongly suggested that SCP-2 mediates cholesterol transport and targeting within the cell (reviewed in Refs. 2 and 3), the advent of newer structural (reviewed in Ref. 4), molecular (reviewed in Refs. 4-6), and gene ablation (7) approaches revealed additional potential roles for SCP-2 in cellular fatty acid metabolism. However, resolving the relative roles of SCP-2 in cholesterol versus fatty acid metabolism has been complicated by the fact that neither the function of the 20-amino acid presequence in 15-kDa pro-SCP-2, the primary SCP-2 gene product, nor how it affects the intracellular targeting of this protein are yet known. Further, little is known about the structure and function pro-SCP-2 because it is posttranslationally completely cleaved to the mature SCP-2 in all tissues examined (reviewed in Ref. 2). In addition, pro-SCP-2 is completely (8) or nearly completely cleaved to SCP-2 in cells transfected with the cDNA encoding pro-SCP-2 (9, 10).

SCP-2 was originally isolated from liver and other tissues as a 13-kDa soluble protein whose amino acid sequence did not identify any consensus sequences targeting SCP-2 to specific intracellular organelle(s), suggesting a primarily cytosolic localization (11, 12). In contrast, cDNA sequencing revealed that the two SCP-2 gene products, 58-kDa SCP-x and 15-kDa pro-SCP-2, as well as the mature 13-kDa SCP-2 contained an C-terminal SKL peroxisomal targeting sequence, thereby suggesting an exclusive peroxisomal localization (reviewed in Refs. 2 and 13). However, immunogold electron microscopy and immunofluorescence imaging showed that, whereas SCP-x appears almost exclusively peroxisomal, the intracellular localization of SCP-2 is more complex with over half of the total SCP-2 being extraperoxisomal where it is localized diffusely in the cytoplasm as well as associated with endoplasmic reticulum and mitochondria (reviewed in Refs. 2, 3, and 13). The fact that the SCP-x and SCP-2 did not copurify with peroxisomes (catalase) in subcellular fractionation further supported differential intracellular distribution of SCP-2 in the cell.

These studies suggest that the 20-amino acid presequence of the pro-SCP-2 may function in some manner to modify the intracellular targeting of this gene product and thereby account for dual functions of SCP-2 gene products in both fatty acid and sterol metabolism. Consistent with this possibility, transfection of cells with the cDNA encoding pro-SCP-2 (8-10, 14, 15) alters cholesterol and/or fatty acid uptake and intracellular diffusion or trafficking (4, 9, 15) differently from that observed on transfection of cells with cDNA encoding the 13-kDa SCP-2 (5, 8, 14). Furthermore, in SCP-2 gene ablated mice (missing all SCP-2 gene products), not only is peroxisomal fatty acid oxidation deranged, liver cholesterol ester and triacylglycerol contents are also decreased 2-fold (7) whereas cholesterol oxidation to bile acids is dramatically reduced (16, 17). These findings are consistent with a potential role for SCP-2 in both peroxisomal and extraperoxisomal cholesterol and/or fatty acid metabolism.

In summary, except for the 20-amino acid N-terminal presequence present in the pro-SCP-2, both pro-SCP-2 and SCP-2 are identical and contain the same C-terminal SKL peroxisomal targeting sequence. The purpose of the present investigation was to determine the potential role(s) of the 20-amino acid presequence of pro-SCP-2 in modulating the structure, function, and intracellular targeting of SCP-2. The latter question gains additional importance since analysis of the pro-SCP-2 20-amino acid N-terminal presequence by a recognition program (e.g. PSORT II) reveals 89% similarity and 83% identity to mitochondrial targeting sequences.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Dehydroergosterol was synthesized and purified (18). cis-Parinaric acid was from Calbiochem (La Jolla, CA). trans-Parinaric acid was from Molecular Probes (Eugene, OR). Carboxypeptidase A, catalase, luciferase, acyl-CoA oxidase, alkaline phosphatase, and FITC conjugates of goat anti-rabbit as well as rat IgG were from Sigma. Rabbit and rat polyclonal anti-13-kDa SCP-2 antibodies were obtained as described earlier (13). Rabbit polyclonal antisera against peptides to the 70-kDa peroxisomal membrane protein (anti-PMP-70) and against a 12-amino acid synthetic peptide (NH2-CRYHLKPLQSKL-COOH) containing the C-terminal SKL peroxisomal targeting sequence 1 (PTS 1) were from Zymed Laboratories Inc. (San Francisco, CA). Texas Red goat anti-rabbit IgG conjugate was from Southern Biotechnology Associates (Birmingham, AL). Nitrocellulose membrane was purchased from Schleicher & Schuell, Sigma, and Bio-Rad.

Pro-SCP-2 and SCP-2 Protein Isolation-- Recombinant human pro-SCP-2 and SCP-2 were isolated as described (19), quantitated by Bradford assay (20). Cholesterol transfer in vitro (21, 22) and in transfected cells (4, 6, 8, 14, 15) indicated that the recombinant as well as transfected cell gene products were properly folded.

Anti-SCP-2 Antibodies, Western Blotting, Dot Blotting, and Immunolabeling-- Polyclonal antisera to SCP-2 were prepared in female New Zealand White rabbits and rats (Hazleton Research Products, Denver, PA) according to the protocols for the use of laboratory animals approved by the appropriate institutional review committee and met Association for Assessment and Accreditation of Laboratory Animal Care guidelines basically as described previously (23). Rabbit polyclonal antibodies were further purified by chromatography on protein A-Sepharose followed by incubation with mouse liver and L-cell homogenates from which all SCP-2 gene products had been removed. This eliminated cross-reactivity to other proteins (24, 25). Specificity of the purified anti-SCP-2 antibodies was determined by quantitative Western blotting (13). Dot blotting was done similarly, except that native protein was spotted directly on the nitrocellulose membrane, dried, and processed as above. Polyclonal rabbit and rat anti-SCP-2 or anti-SKL peptide were primary antibodies. Bands of interest were visualized with alkaline-phosphatase conjugates of secondary antibodies and nitro blue tetrazloium/5-bromo-4-chloro-3-indolyl phosphate substrate (Sigma). For immunofluorescence studies (see below), the working dilution of purified anti-13-kDa SCP-2 antibody was 1:20 and 1:40 (for double labeling). FITC- and Texas Red (for double labeling)-conjugated goat anti-rabbit and anti-rat IgG were used as secondary antibodies at (1:100) (Sigma and Southern Biotechnology Associates, Birmingham, AL).

Circular Dichroism-- Circular dichroic spectra of recombinant human pro-SCP-2 and SCP-2 (2 µM) dissolved in 10 mM phosphate buffer, pH 7.4, were recorded as described (26). Secondary structure analysis was performed with the program CDsstr (27, 28) using a singular value decomposition algorithm described earlier (29). This analysis provided estimation of five protein secondary structure components: alpha -helix, 310-helix, beta -strand, beta -turn, poly(L-proline)II type 31-helix, and other. Guanidine-HCl unfolding as well as determination of (i) the guanidine concentration for 50% unfolding, (ii) the observed free energy change Delta Gobs, and (iii) the Gibbs free energy change Delta G0 in the absence of denaturant were performed as described earlier (30).

Mass Spectroscopy of Pro-SCP-2 and SCP-2 Proteolyzed by Carboxypeptidase A-- Carboxypeptidase A was diluted to a concentration of 2.8 58 M in 25 mM NH4HCO3, 1 mM ZnCl2, pH 8. Stock solutions of SCP-2 and pro-SCP-2 in 10 mM potassium phosphate, 1 mM ZnCl2, pH 8, were diluted to the concentration of interest (0.1-1.1 mg/ml) in 10 mM potassium phosphate, 1 mM ZnCl2, pH 8. Carboxypeptidase A initiated the reaction, and samples were sequentially collected, injected into 1.5 µl of matrix solution (100 mM ferulic acid, 0.1% trifluoroacetic acid, methanol) to quench the reaction, and spotted onto gold-plated target plates precoated with ferulic acid (500 mM). Masses were analyzed using a Voyager Elite XL time-of-flight mass spectrometer equipped with delayed extraction (PerSeptive Biosystems, Framingham, MA): acceleration voltage 25 KeV, 93.5% grid voltage, 0.035% guide wire voltage, and a delay of 250 ns. Mass peak areas were determined by Grams (Galactic Industries Corp.), plotted versus time, and analyzed using Excel (Microsoft Corp.).

Ligand Binding and Sterol Exchange-- NBD-cholesterol binding to 15-kDa and 13.2-kDa SCP-2 were determined at 25 °C (31) as described earlier (32), except that SCP-2 was 11 nM (10 mM phosphate buffer, pH 7.4) and NBD-cholesterol was added in 0.5-2.0 µl of dimethylformamide. Parinaric acid binding was done as described (32-34). Fluorescent sterol exchange assays were done at 37 °C as described previously (35, 36).

Indirect Immunofluorescence Microscopy-- L-cells (L-arpt-tk-) and L-cells transfected with cDNAs encoding for pro-SCP-2 or SCP-2 (8) were cultured in 10% fetal bovine serum Higuchi medium (37). McA-RH7777 hepatoma cells transfected with the cDNA encoding pro-SCP-2 were cultured as described (9). Rabbit and rat anti-13-kDa SCP-2 polyclonal antibodies were the primary antibodies for detecting SCP-2 in the cells. Peroxisomes were localized with polyclonal affinity purified sheep anti-bovine liver catalase (BioDesign International, Kennebunk, ME), as well as with rabbit anti-PMP70 antiserum (Zymed Laboratories Inc., San Francisco, CA). The secondary antibodies used were: FITC-conjugated donkey anti-sheep IgG (Sigma), Texas Red-conjugated goat anti-rabbit IgG (SBA, Birmingham, AL), and FITC-conjugated goat anti-rat IgG (Sigma). Cells were grown on glass coverslip chambers and prepared for indirect immunofluorescence as described earlier (6, 13). Laser scanning confocal microscopy, immunocolocalization, and image analysis were performed as described earlier (6, 13, 21, 22).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Secondary Structure of Pro-SCP-2 and SCP-2 Determined by Circular Dichroism-- As indicated in the Introduction, there are no available data explaining the differential effects of expressing the pro-SCP-2 versus SCP-2 on cholesterol and fatty acid uptake, intracellular trafficking, and metabolism in transfected cells. If these proteins differed in secondary structure conformation, this could affect their relative targeting to peroxisomes via their C-terminal SKL peroxisomal targeting sequences. However, other than its cDNA-derived primary amino acid sequence, nothing is known regarding secondary or tertiary structure of pro-SCP-2. Therefore, the secondary structure of pro-SCP-2 and SCP-2 were determined by circular dichroism.

The circular dichroic spectra of pro-SCP-2 and SCP-2 differed dramatically. SCP-2 circular dichroic spectra displayed strong minima at 208 and 225 nm and one maximum at 193 nm, consistent with the presence of significant alpha -helical secondary structure (Fig. 1A, triangles). In contrast, pro-SCP-2 circular dichroic spectra (Fig. 1A, squares) were attenuated 4-5-fold in intensity and were more asymmetrical in shape as compared with that of SCP-2. Quantitative analysis of the spectra, determined as described under "Experimental Procedures," provided a high accuracy assessment for the following secondary structures: alpha -helix (H), 310-helix (G), beta -strand (E), turn (T), and poly(L-proline)II type 31-helix (P). All other structures unresolved by this algorithm are designated as "other" (O). The experimental and theoretical fits of the spectra were nearly superimposable (data not shown). Pro-SCP-2 had less than 20% helical structure (H + G + P in Fig. 1B, black bars). The predominant components in the secondary structure of pro-SCP-2 were beta -turn and beta -strand, which represent about 40% of its structure (E + T in Fig. 1C, black bars). In marked contrast, SCP-2 was predominantly helical structured, with the total helix fractions being approximately 50% (H + G + P in Fig. 1B, shaded bars) and only about 20% beta -structure (E + T in Fig. 1C, shaded bars). Both proteins contained about the same fraction of unresolved structures (O in Fig. 1C). In summary, the presence of the N-terminal 20-amino acid presequence in pro-SCP-2 dramatically altered the secondary structure.


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  		Fig. 1.   Circular dichroism and secondary structure analysis of 15-kDa pro-SCP-2 and 13-kDa SCP-2. Panel A, circular dichroism spectra of pro-SCP-2 (squares) and SCP-2 (triangles) (3 µM) in phosphate buffer, pH 7.4, 25 °C. For more details, see "Experimental Procedures." Secondary structure analysis was obtained according to an algorithm (27, 28). Panel B, the helical component of 15-kDa pro-SCP-2 (black bars) and 13-kDa SCP-2 (gray bars) secondary structure, where H is alpha -helix, G is 310-helix, and P is poly(L-proline)II type 31-helix. Panel C, the beta -structure component of 15-kDa (black bars) and 13.2-kDa (gray bars) SCP-2, where E is beta -strand, T is beta -turn, and O is the other structures.

Stability of Pro-SCP-2 and SCP-2 to Denaturation-- The relative stability of 15-kDa pro-SCP-2 and 13-kDa SCP-2 to guanidine-HCl-induced unfolding was determined by monitoring the reduction of circular dichroism at 222 nm, as indicated under "Experimental Procedures." At increasing guanidine-HCl, the negative molar ellipticity [theta ]225 of SCP-2 (Fig. 2A) and pro-SCP-2 (Fig. 2B) was markedly reduced. The concentration of guanidine-HCl required for 50% unfolding, 0.80 and 0.82 M, respectively, did not differ between the two proteins. Plots of the observed free energy change Delta Gobs versus guanidine-HCl concentration for both proteins were linear (Fig. 2, A (inset) and B (inset)) and allowed calculation of the Gibbs free energy change Delta G0 in the absence of denaturant, 14.7 and 14.9 kJ/mol, respectively, for the two proteins. These data indicate that pro-SCP-2 and SCP-2 did not differ in stability.


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  		Fig. 2.   Guanidine-HCl-induced unfolding of SCP-2 and pro-SCP-2. Guanidine-HCl-induced denaturation of SCP-2 (panel A) and pro-SCP-2 (panel B) was performed at 24 °C by measuring molar ellipticity at 222 nm by circular dichroism as described under "Experimental Procedures." The concentration of each protein, determined from ultraviolet spectra (see "Experimental Procedures") was maintained at 4 µM. The solid lines represent the fitted curves, and the dotted lines represent pre- and post-unfolding base lines.

Pro-SCP-2 and SCP-2 Tertiary Structure Examined by Surface Exposure of Epitopes to Polyclonal Anti-13-kDa SCP-2 Antisera-- Since circular dichroism revealed that the presence of the 20-amino acid N-terminal presequence dramatically altered the secondary structure of the protein, it was important to determine if this also resulted in modified tertiary structure. Polyclonal antisera against the SCP-2 reacted similarly with unfolded pro-SCP-2 and SCP-2 as determined by SDS-PAGE followed by Western blotting (data not shown). In contrast, when the proteins were examined by dot blotting, they differed markedly (Fig. 3). Visual examination of the dot blots revealed that the anti-SCP-2 antisera reacted more strongly at all concentrations tested with SCP-2 as compared with equimolar pro-SCP-2. This effect was even more pronounced than observed with the SCP-x (Fig. 3), the other SCP-2 gene product whose C-terminal 13-kDa polypeptide is identical to SCP-2. These differences in accessibility of the surface epitopes of SCP-2 and pro-SCP-2 were not likely due to differences in unfolding on preparation of the dot blots because these proteins exhibited nearly identical stability (Fig. 2, A and B).


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  		Fig. 3.   Dot blots of SCP-2 gene products incubated with anti-13-kDa SCP-2. Representative dot blots of equimolar aliquots (1, 10, 100 µM) of 13-kDa SCP-2, 15-kDa pro-SCP-2, and 58-kDa SCP-x on nitrocellulose in the absence of denaturing detergents were developed with antibodies as described under "Experimental Procedures."

Exposure of the C-terminal SKL of Pro-SCP-2 and SCP-2 to Antisera against a Peptide Containing the C-terminal SKL-- In order to more specifically determine the relative surface exposure of the C-terminal SKL peroxisomal targeting sequence, SCP-2 gene products as well as several other known peroxisomal proteins were probed with commercially available antisera generated against a peptide (NH2-CRYHLKPLQSKL-COOH) containing the C-terminal SKL peroxisomal targeting sequence 1 (PTS 1). The last 9 amino acids of this peptide corresponded to the C terminus of rat peroxisomal proteins (acyl-CoA oxidase and hydratase-dehydrogenase bifunctional enzyme) (38). Consistent with previous reports (38-41), anti-SKL antibodies detected multiple peroxisomal proteins in liver (Fig. 4, top panel). SDS-PAGE and Western blotting were performed on equimolar amounts of known peroxisomal proteins: acyl-CoA oxidase (C-terminal 9 amino acids identical to the SKL peptide to which the antibodies were generated) and catalase and luciferase (sharing only the C-terminal 3 amino acids SKL with the SKL peptide to which the antibodies were generated). Although the anti-SKL antibodies detected all three unfolded peroxisomal proteins (Fig. 4, top panel), they differentially reacted with these unfolded proteins in the order: luciferase [tmt] catalase > acyl-CoA oxidase (Fig. 4, top panel). When the anti-SKL reactivity of these proteins was examined in the folded state by dot blotting (Fig. 4, bottom panel), they reacted essentially equally well as in the unfolded state (Fig. 4, top panel) and in the quantitative order: luciferase >>> catalase > acyl-CoA oxidase (not shown). These data with unfolded, as well as folded, peroxisomal proteins suggest that the large difference in reactivity was most likely due to differences in their amino acid sequences adjacent to the C-terminal three SKL amino acids.


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  		Fig. 4.   Reactivity of unfolded and folded C-terminal SKL containing proteins with anti-SKL antibodies. Top panel, SDS-PAGE followed by Western blot. Bottom panel, dot blot. Quantitative densitometry was performed on dot blots containing equimolar amounts of proteins with C-terminal SKL following probing with anti-SKL antibodies: luciferase, acyl-CoA oxidase, catalase, 13-kDa SCP-2, 15-kDa pro-SCP-2, and 58-kDa SCP-x.

In contrast to the above proteins, which arise from different genes, the SCP-2 gene products (13-kDa SCP-2, 15-kDa pro-SCP-2, and 58-kDa SCP-x) contain the identical C-terminal 13-kDa SCP-2 polypeptide chain. Pro-SCP-2 and SCP-2 reacted equally well on Western blots (unfolded) with anti-SKL antibodies, whereas SCP-x reacted poorly with anti-SKL antibodies, either as pure protein or in the liver homogenate (Fig. 4, top panel). In contrast, these proteins reacted much less strongly in dot blots (folded) (Fig. 4, bottom panel) as compared with the unfolded state (Fig. 4, top panel), but nevertheless still in the order: pro-SCP-2 = SCP-2 >>> SCP-x (Fig. 4, bottom panel). In summary, the C-terminal SKL peroxisomal targeting tripeptide was poorly exposed at the surface of the SCP-2 family of proteins in the folded state and anti-SCP-2 IgG, with their large size (150 kDa), did not readily access these C-terminal SKL tripeptides in the folded proteins to detect specific differences as compared with other peroxisomal proteins.

Surface Exposure of the C-terminal Leucine Residue Present in Pro-SCP-2 and SCP-2-- Since the large size (e.g. 150 kDa) of IgGs may interfere with detecting differences in surface exposure of the C-terminal SKL of pro-SCP-2 and SCP-2, the relative reactivity of the two proteins to the much smaller (5-fold lower molecular mass than 150-kDa IgG) carboxypeptidase A was examined. Carboxypeptidase cleaves only the C-terminal Leu, as shown by matrix-assisted laser desorption-induced/time-of-flight (MALDI-TOF) mass spectroscopy. With increasing carboxypeptidase A incubation time (Fig. 5A, curves a-i), a new mass peak at 113-Da lower mass appeared, consistent with the loss of the C-terminal Leu. Cleavage of the C-terminal Leu from pro-SCP-2 was nearly 6-fold faster as compared with SCP-2 (Fig. 5B). Lineweaver-Burke plots (data not shown) revealed that the Km, Vmax, and kcat for pro-SCP-2 (13.1 × 10-10, 9.5 × 10-11, and 34.4) differed from those of SCP-2 (7.5 × 10-10, 5.4 × 10-11, and 19.6). Thus, the C-terminal Leu (i) was exposed at the surface of both pro-SCP-2 and SCP-2, and (ii) that of pro-SCP-2 was more accessible to cleavage by carboxypeptidase A. 


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  		Fig. 5.   MALDI-TOF mass spectra of SCP-2 treated with carboxypeptidase A. Panel A, SCP-2 (1.1 mg/ml) was treated with carboxypeptidase A for varying times as described under "Experimental Procedures," followed by MALDI-TOF mass spectroscopic analysis. The mass pertaining to SCP-2 appears at approximately 13,226 Da, whereas that of the product of the reaction appears at approximately 13,100 Da. Spectrum a represents the initial reaction mixture after 1 min, b = 2 min, c = 3 min, d = 4 min, e = 5 min, f = 8 min, g = 19 min, h = 30 min, i = 13.5 h. Panel B, time course of the development of the carboxypeptidase A product of SCP-2 and pro-SCP-2. SCP-2 (squares, bottom curve) and pro-SCP-2 (diamonds, top curve) mg/ml were reacted with carboxypeptidase A as described under "Experimental Procedures." p/[p + r] is defined as the rate of C-terminal Leu cleavage by carboxypeptidase A. The inset in panel B shows an expanded region (same x and y axes) at the beginning time course.

Effect of the Pro-SCP-2 N-terminal Presequence on Ligand Binding-- Since the above studies showed that pro-SCP-2 and SCP-2 differed in tertiary and, even more markedly, in secondary structure, it was important to determine that both proteins were functionally active and to determine if they differed in activity. Pro-SCP-2 and SCP-2 both bound NBD-cholesterol with 1:1 stoichiometry and similar Kd values, 4.09 ± 2.08 and 4.15 ± 1.42 nM (n = 4), respectively (Table I). Pro-SCP-2 bound cis-parinaric acid (a fluorescent fatty acid with a kinked-carbon chain, typical of naturally occurring unsaturated fatty acids) with 1:1 stoichiometry and Kd = 0.36 ± 0.14 µM (n = 6) (Table I). This Kd for cis-parinaric acid binding was indistinguishable from that for the 13-kDa SCP-2 (Table I) reported previously (32). Pro-SCP-2 did not exhibit simple saturation binding with trans-parinaric acid (a fluorescent, straight chain fatty acid, typical of naturally occurring saturated fatty acids) and Scatchard plots were biphasic with Kd1 = 0.2 ± 0.14 µM and Kd2 = 2.24 ± 0.76 µM (n = 5) (Table I). The presence of two trans-unsaturated fatty acid binding sites in pro-SCP-2 differed markedly from the single site in SCP-2 (Table I), although its affinity was the same as that of the pro-SCP-2 Kd1 site (26, 32).

                              
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Table I
Cholesterol and fatty acid binding to pro-SCP-2 and SCP-2
Values represent the mean ± S.E. (n = 4-6).

Function of the N-terminal Presequence in Pro-SCP-2: Intermembrane Cholesterol Transfer from Plasma Membranes-- Spontaneous sterol transfer from plasma membranes, determined as described under "Experimental Procedures," proceeded slowly as indicated by increasing dehydroergosterol polarization with increasing incubation time (data not shown). The addition of the SCP-2, but not pro-SCP-2, dramatically increased the transfer of dehydroergosterol from plasma membranes (data not shown). Quantitative analysis with the aid of a standard curve (see "Experimental Procedures") allowed conversion of the fluorescence polarization changes to molecular sterol transfer. The initial rate of spontaneous molecular sterol transfer, 0.68 pmol/min, was increased 6-fold by SCP-2, but not pro-SCP-2. When the spontaneous sterol exchange curves were fitted to single and multiple exponentials (see "Experimental Procedures"), only one kinetic component with half-time of 139 min was observed for spontaneous sterol transfer. Addition of SCP-2, but not pro-SCP-2, shortened the half-time of sterol transfer.

Intracellular Targeting of Pro-SCP-2 and SCP-2 in Transfected L-cell Fibroblasts-- As indicated in the Introduction, both pro-SCP-2 and SCP-2 have the same C-terminal SCL peroxisomal targeting sequence. However, since the above data indicated that these proteins differed in secondary and tertiary structure of these proteins, this suggested that the N-terminal 20-amino acid presequence of pro-SCP-2 may modulate the intracellular targeting of SCP-2 to peroxisomes. To resolve this question, L-cells transfected with cDNA encoding the 13-kDa SCP-2 were double immunolabeled with antisera to 13-kDa SCP-2 and to PMP70, a peroxisomal membrane marker, and examined by laser scanning confocal microscopy. A representative 0.3-µm confocal slice through the cell and its nucleus (dark area in middle of slice) is shown in Fig. 6. The intracellular distribution pattern of PMP-70 was punctate (Fig. 6A) and colocalized with that of catalase (data not shown). In contrast, the anti-13-kDa SCP-2 labeling pattern of L-cells expressing the 13-kDa SCP-2 was much less punctate and more diffuse (Fig. 6B). Superposition of the simultaneously acquired fluorescence patterns for peroxisomal PMP-70 (red) and 13-kDa SCP-2 (green) revealed weak colocalization in punctate structures (yellow) with most of the immunofluorescence appearing as separate red and green areas (Fig. 6C). These qualitative data were displayed as a pixel fluorogram (Fig. 6D). Complete colocalization would have yielded strongly yellow pixels all localized along the diagonal of the fluorogram. However, examination of Fig. 6D showed few yellow pixels localized along the fluorogram diagonal. Most of the anti-SCP-2 pixels (green) were distinct from those of peroxisomal PMP-70 (red). The ratio of green pixels colocalizing with red pixels was only 0.14, indicating that only 14% of SCP-2 colocalized in peroxisomes. These results suggested that, in the absence of the N-terminal 20-amino acid presequence, the 13-kDa SCP-2 was very weakly targeted to peroxisomes.


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  		Fig. 6.   Intracellular localization of SCP-2 in L-cells transfected with cDNA encoding 13-kDa SCP-2. Double immunolabeling with rabbit anti-PMP-70 (peroxisomal membrane protein-70) and rat anti-SCP-2 and laser scanning confocal microscopic images was performed as described under "Experimental Procedures." A, anti-PMP-70 detected with Texas Red-conjugated secondary antibody. B, anti-13-kDa SCP-2 detected with FITC-conjugated secondary antibody. C, simultaneously acquired images A and B were superimposed to yield a merged image. Colocalized PMP-70 and 13-kDa SCP-2 appeared as yellow-orange punctate structures. D, pixel fluorogram of the merged image showing the low degree of PMP-70 and 13-kDa SCP-2 fluorescence spatial correlation (red, 0.15; green, 0.14).

The intracellular distribution of SCP-2 in L-cells transfected with the cDNA encoding the 15-kDa pro-SCP-2 differed significantly from that of cells transfected with cDNA encoding 13-kDa SCP-2, even though Western blotting revealed that the 15-kDa pro-SCP-2 is completely post-translationally processed to 13-kDa SCP-2 in L-cells, i.e. no detectable 15-kDa pro-SCP-2 on Western blots (8). In the L-cells transfected with cDNA encoding pro-SCP-2, the intracellular distribution pattern of PMP-70 was still punctate (Fig. 7A) whereas that of SCP-2 was again both punctate and diffuse (Fig. 7B). However, superposition of these simultaneously acquired fluorescence patterns revealed more extensive colocalization in punctate structures (yellow) (Fig. 7C) than observed with L-cells transfected with cDNA encoding 13-kDa SCP-2 (Fig. 7C). This qualitative analysis was quantitatively confirmed by comparison of the pixel fluorograms (Fig. 7D versus Fig. 6D). The ratio of green pixels colocalizing with red pixels was 0.45 (Fig. 7D), more than 3-fold higher in 15-kDa pro-SCP-2 transfectants than with 13-kDa SCP-2 transfectants (Fig. 6D). However, in either case more than 50% of SCP-2 was localized outside peroxisomes in L-cells.


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  		Fig. 7.   Intracellular targeting of SCP-2 in L-cells transfected with cDNA encoding the 15-kDa pro-SCP-2. Western blots of these cells showed only 13-kDa SCP-2, with no detectable 15-kDa pro-SCP-2 (8). Double immunolabeling with rabbit anti-PMP-70 (peroxisomal membrane protein-70) and rat anti-13-kDa SCP-2 followed by simultaneous acquisition of laser scanning confocal microscopic images was performed as described under "Experimental Procedures." A, anti-PMP-70 was detected with Texas Red-conjugated secondary antibody. B, anti-SCP-2 was detected with FITC-conjugated secondary antibody. C, simultaneously acquired images A and B were superimposed to yield a merged image. Colocalized PMP-70 and SCP-2 appeared as yellow-orange punctate structures. D, pixel fluorogram of the merged image showing the higher degree of PMP-70 and SCP-2 fluorescence spatial correlation (red, 0.31; green, 0.45).

Intracellular Targeting of Pro-SCP-2 in Transfected Hepatoma Cells-- It is possible that the intracellular targeting of pro-SCP-2, the primary SCP-2 gene product, may be based on the cell type that pro-SCP-2 was transfected into. Therefore, the intracellular localization of SCP-2 was examined in McA-RH777 hepatoma cells transfected with cDNA encoding pro-SCP-2 (9).

Quantitative densitometry of Western blots of control and mock transfected hepatoma cells (9) showed that these cells expressed similar very low levels of 13-kDa SCP-2 (10-20-fold lower than in liver). However, the 15-kDa pro-SCP-2 was undetectable in either control or mock-transfected hepatoma cells. Control hepatoma cells were fixed and double immunolabeled to detect the peroxisomal marker PMP70 (red) and SCP-2 (green), followed by simultaneous colocalization of the two proteins by laser scanning confocal fluorescence microscopy. Superposition of the two images (Fig. 8A) showed primarily a red (PMP70 label), punctate pattern typical of peroxisomes. As expected from the Western blots, almost no green (SCP-2) areas were visible. This was quantitatively confirmed in the pixel fluorogram (Fig. 8B). The pixel fluorogram showed a population of red pixels (PMP70) along the y axis that quantitatively showed almost no overlap with green (SCP-2). The small amount of detectable SCP-2 was localized primarily along the x axis with 58% of this SCP-2 localized in peroxisomes. Nearly identical data (data not shown) were obtained with mock-transfected hepatoma cells.


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  		Fig. 8.   Intracellular targeting of SCP-2 in McA-RH7777 hepatoma cells transfected with cDNA encoding the 15-kDa pro-SCP-2. Double immunolabeling with rabbit anti-PMP-70 (peroxisomal membrane protein-70) and rat anti-SCP-2 followed by simultaneous acquisition of laser scanning confocal microscopic images was performed as described under "Experimental Procedures." A, superimposed confocal image of anti-PMP-70 (Texas Red-conjugated secondary antibody) and anti-SCP-2 (FITC-conjugated secondary antibody) in control McA-RH7777 hepatoma cells. B, pixel fluorogram of data in panel A. C, superimposed confocal image as in panel A except that the image was of a transfected McA-RH7777 hepatoma clone in which 95% of pro-SCP-2 was cleaved to SCP-2. D, pixel fluorogram of panel C. E, superimposed confocal image as in panel A except that the image was of another transfected McA-RH7777 hepatoma clone in which 60% of pro-SCP-2 was cleaved to SCP-2. F, pixel fluorogram of panel E. In panels A, C, and E, the colocalized PMP-70 and SCP-2 appeared as yellow-orange punctate structures. Pixel fluorograms showed high degree of SCP-2 spatial correlation with PMP-70; green was 0.58, 0.59, and 0.59 in the control and two transfected hepatoma clones, respectively.

In transfected hepatoma cells expressing immunoreactive SCP-2 gene products at levels essentially the same as in liver, only 5% of total SCP-2 was uncleaved 15-kDa pro-SCP-2 (9). Simultaneous colocalization of the PMP-70 and SCP-2 by laser scanning confocal fluorescence microscopy and superposition of the two images (Fig. 8C) showed a significant amount of colocalization (yellow/orange punctate areas), but also separate populations of red and green areas. A pixel fluorogram (Fig. 8D) showed a colocalizing population of the SCP-2 with peroxisomal marker designated as yellow/orange pixels along the diagonal. However, distinct populations of red and green pixels showed that significant amount of SCP-2 did not colocalize with peroxisomes. Quantitative analysis of the pixel fluorogram showed that 59% of SCP-2 colocalized with the peroxisomal marker.

In transfected hepatoma cells expressing immunoreactive gene products at 2.2-fold higher levels than in liver showed 40% of total SCP-2 was uncleaved 15-kDa pro-SCP-2 (9). Colocalization of the PMP-70 and SCP-2 by laser scanning confocal fluorescence microscopy and superposition of the two images (Fig. 8E) again showed a significant amount of colocalization (yellow/orange punctate areas), but also separate populations of red and green areas. A pixel fluorogram (Fig. 8F) confirmed this colocalization of SCP-2 with peroxisomal marker (yellow/orange pixels) located along the diagonal. However, the appearance of distinct populations of red and green pixels showed that significant amount of SCP-2 did not colocalize with peroxisomes. Quantitative analysis of the pixel fluorogram showed that, despite the fact that 40% of pro-SCP-2 was not cleaved to SCP-2 (9) 59% of SCP-2 was still colocalized with the peroxisomal marker (Fig. 8F).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There is a curious disparity between the size the SCP-2 gene product on Western blotting as compared with analysis of the cDNA. Western blots of all tissues and primary cultured cells examined (reviewed in Ref. 2) reveal only the 13-kDa form of SCP-2. In contrast, the SCP-2 gene encodes a larger 15-kDa pro-SCP-2 protein product containing a 20-amino acid N-terminal presequence. This suggests that the 15-kDa pro-SCP-2 is posttranslationally cleaved to the mature 13-kDa SCP-2. In cells transfected with cDNA encoding the 15-kDa pro-SCP-2, the degree of cleavage to the mature 13-kDa SCP-2 was dependent on both cell type and the level of 15-kDa pro-SCP-2 expression. In L-cells transfected with cDNA encoding 15-kDa pro-SCP-2, only the 13-kDa SCP-2 is detectable (8). In transfected COS-7 cells (9) and intermediate expression McA-RH7777 hepatoma cells (9), a small amount of 15-kDa pro-SCP-2 was detectable (about 5% of total SCP-2 in the hepatoma cells). In contrast, in transfected McA-RH7777 hepatoma cells expressing 15-kDa pro-SCP-2 at levels higher than in liver, as much as 40% immunoreactivity is present as uncleaved 15-kDa pro-SCP-2 whereas 60% was cleaved to 13-kDa SCP-2 (9). This suggests that the 15-kDa pro-SCP-2 is posttranslationally cleaved to the mature 13-kDa SCP-2.

Although transfected cell data are suggestive that the N-terminal presequence modifies the role of SCP-2 in lipid metabolism (4, 5, 8-10, 14, 15), almost nothing is known regarding the mechanism whereby this occurs. There have been no reports detailing the effects of the presequence on the secondary or tertiary structure of SCP-2. Neither are there definitive data elucidating if this presequence alters the function of SCP-2 either in lipid binding/lipid transfer from biological membranes or in intracellular targeting of SCP-2. The data presented herein provide several new insights regarding these issues.

First, the results directly demonstrated for the first time that the 20-amino acid presequence dramatically alters the secondary structure of SCP-2. Circular dichroism showed that 15-kDa pro-SCP-2 exhibited 2.5-fold lower percentage of helical structure and 2-fold more beta -structure as compared with the 13-kDa SCP-2. The tertiary structure of 15-kDa pro-SCP-2 (probed by anti-SCP-2 antisera, anti-SKL antisera, and carboxypeptidase A/MALDI-TOF mass spectroscopy) showed marked differences in the surface exposure of epitopes and the C-terminal Leu as compared with 13-kDa SCP-2. The altered secondary and tertiary structure of pro-SCP-2 were not due to altered stability or functional inactivity.

Second, the N-terminal 20-amino acid presequence modified SCP-2 function in vitro. The 15-kDa pro-SCP-2 exhibited two fatty acid binding sites as compared with only one fatty acid binding site for SCP-2. Furthermore, the ability of SCP-2 to mediate sterol transfer from plasma membranes was markedly decreased by the presence of the 20-amino acid presequence despite the fact that both proteins had equal affinity for cholesterol binding. The presequence also inhibited sterol transfer from mitochondrial and lysosomal membranes (21, 22). Earlier data from this laboratory also showed that SCP-2 has not only a ligand binding site (reviewed in Ref, 2), but also a membrane interaction site located at the N terminus (42-44). Membrane interaction enhanced SCP-2 mediated cholesterol transfer (44). This explains why the presence of the N-terminal presequence in pro-SCP-2 inhibited the ability of the protein to mediate sterol transfer from biological membranes without affecting sterol binding.

Third, the immunolocalization results with transfected L-cells and hepatoma cells helped to clarify one of the major controversies in the SCP-2 field, i.e. whether the SCP-2 is exclusively targeted to peroxisomes? The data clearly demonstrated that the C-terminal SKL peroxisomal targeting sequence in 13-kDa SCP-2 was not in itself sufficient to mediate complete targeting of the protein to peroxisomes. In cells transfected with cDNAs encoding either the 13-kDa SCP-2 (or the 15-kDa pro-SCP-2), the majority of anti-SCP-2 immunolabeling was not associated with peroxisomes. Only 45% of SCP-2 was localized with peroxisomes in L-cells transfected with the cDNA encoding 15-kDa pro-SCP-2. Similarly 59% of SCP-2 in hepatoma cells transfected with cDNA encoding pro-SCP-2 was localized to peroxisomes (Fig. 8). Since the distribution of SCP-2 in peroxisomes was the same in transfected hepatoma cells wherein 5% and 40% of pro-SCP-2 was not cleaved to 13-kDa SCP-2, these data suggest that the amount (but not percentage of distribution) of pro-SCP-2 in peroxisomes was dependent on the level of pro-SCP-2 expressed. However, it must be noted that the exact location of the 15-kDa pro-SCP-2 in the transfected hepatoma cells awaits the availability of antisera specific for the N-terminal 20 amino acids. In summary, these data indicated that in L-cells as well as multiple liver-derived cell lines SCP-2 was not exclusively peroxisomal and that significant extraperoxisomal cleavage of pro-SCP-2 occurred. This suggestion was supported by observation that 58-kDa SCP-x, but not 13-kDa SCP-2, copurifies with peroxisomal markers (45-47)and that SCP-2 expression enhances many extraperoxisomal functions (e.g. fatty acid uptake, intracellular fatty acid diffusion, and fatty acid incorporation into cholesterol esters via ACAT in the endoplasmic reticulum) (4, 14, 15). Although the structural basis for the weak peroxisomal targeting of the 13-kDa SCP-2 is not entirely clear, the data presented herein suggested at least two possibilities. (i) The folding of the 13-kDa SCP-2 polypeptide chain allows for poor surface exposure of the C-terminal SKL. This possibility is consistent with earlier studies wherein polyclonal anti-13-kDa SCP-2 reacted with three tryptic peptides encompassing amino acid residues 68-108 in 13-kDa SCP-2, but not with peptides encompassing the C-terminal amino acids of 13-kDa SCP-2 (11). (ii) The 13-kDa SCP-2 may be proteolytically modified in the cell or tissue to disrupt the C-terminal SKL peroxisomal targeting sequence. Earlier reports showed that the majority (82-88%) of native 13-kDa SCP-2 isolated from rat or bovine liver did not contain a C-terminal Leu, i.e. the C-terminal SKL peroxisomal targeting sequence was disrupted (11, 12). Interestingly, this percentage of native SCP-2 missing the C-terminal Leu was identical to the percentage (88%) of SCP-2 shown by immunofluorescence imaging to be extraperoxisomal in L-cells transfected with cDNA encoding the 13-kDa SCP-2 (see data shown herein). An intact C-terminal SKL is essential for targeting to SCP-2 peroxisomes, as shown by 3T3 cells microinjected with fluorescent labeled pro-SCP-2 from which the C-terminal Leu is removed. The anti-SCP-2 immunofluorescence pattern in these cells was primarily diffuse (extraperoxisomal) (48). Either or both of the above two explanations were consistent with the observation that 13-kDa SCP-2 was only poorly targeted to L-cell peroxisomes.

Fourth, the data for the first time demonstrated the functional importance of the N-terminal 20-amino acid presequence present in the 15-kDa pro-SCP-2 for efficient intracellular targeting of the SCP-2 to peroxisomes. In L-cells transfected with the cDNA encoding the 15-kDa pro-SCP-2, 3-fold more SCP-2 was targeted to peroxisomes as compared with cells transfected with cDNA encoding 13-kDa SCP-2 (compare Figs. 6 and 7). Likewise, immunofluorescence (Fig. 8) and immunogold electron microscopy (9) revealed efficient targeting of SCP-2 to peroxisomes in hepatoma cells transfected with the cDNA encoding 15-kDa pro-SCP-2. However, in both L-cells and hepatoma cells, less than 60% of SCP-2 was peroxisomal. The available evidence suggests that the N-terminal cleavage of 15-kDa pro-SCP-2 is dependent on tissue/cell type as well as the level of total expression. The 15-kDa pro-SCP-2 is completely cleaved to 13-kDa SCP-2 in all tissues examined (reviewed in Ref. 2) as well as in transfected L-cells expressing SCP-2 at a level near half that detected in liver (8). In transfected hepatoma cells expressing levels of SCP-2 similar to liver, 95% of 15-kDa pro-SCP-2 was cleaved (9). In contrast, only 60% was cleaved when transfected hepatoma cells expressed SCP-2 at levels 2.2-fold higher than in liver (9). These data as well as the immunolocalization presented herein suggest that significant cleavage of the 15-kDa pro-SCP-2 N-terminal 20-amino acid presequence occurs outside the peroxisome. Thus, the stability of the SCP-2 gene product N-terminal amino extension to N-terminal proteolytic cleavage in the cytoplasm may influence the efficiency of targeting the protein to peroxisomes. Consistent with this possibility, recent data showed that 58-kDa SCP-x, a protein exclusively localized to peroxisomes, was stably expressed in the cytoplasm but not cleaved in cells lacking peroxisomes, i.e. Zellweger's fibroblasts (17). In contrast, there was no detectable 15-kDa pro-SCP-2 in the peroxisome-deficient Zellweger's fibroblasts (49). Thus, the longer 45-kDa N-terminal extension in 58-kDa SCP-x protected the protein from proteolytic cleavage outside the peroxisome and conferred even more efficient targeting to peroxisomes as compared with the much shorter 2-kDa N-terminal extension present in 15-kDa pro-SCP-2. In conclusion, these observations contribute significantly to our understanding of the role of the N-terminal 20-amino acid presequence encoded by the SCP-2 gene, but not observed in tissues. The less than 100% efficient targeting of 15-kDa pro-SCP-2 to peroxisomes may account for the ability of this protein to enhance many aspects of extraperoxisomal lipid transfer and metabolism including: cholesterol uptake, plasma membrane cholesterol traffic to the endoplasmic reticulum, lipid droplet cholesterol traffic to mitochondria, fatty acid uptake, intracellular fatty acid diffusion, fatty acid esterification in the endoplasmic reticulum, etc. (4, 8-10, 14, 15). In contrast, the function of peroxisomal SCP-2 has not yet been resolved.

In summary, the present findings indicate that the N-terminal 20-amino acid presequence of the 15-kDa pro-SCP-2 dramatically altered the secondary and tertiary structure of SCP-2, its function in ligand binding/transfer in vitro, and its efficiency for targeting to peroxisomes in intact cells. However, since peroxisomes account for less than 1% of cell protein (reviewed in Ref. 2), quantitatively most of the SCP-2 is extraperoxisomal. This finding was consistent with immunogold labeling studies which showed that, although the highest concentration of anti-SCP-2 immunolabeling (reacting to both 58-kDa SCP-x and SCP-2) was in the peroxisome, the peroxisomes accounted for only a small part of cell protein and cell volume (reviewed in Ref. 2). In conclusion, these observations contribute significantly to our understanding of the role of the N-terminal 20-amino acid presequence.

    ACKNOWLEDGEMENT

We appreciate the helpful assistance of Dr. Hsu Chao in analysis of the 15-kDa pro-SCP-2 by the PSORT II recognition program.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants GM 31561 and DK41402 (to F. S. and A. B. K.) and P30-ES0916 (to A. B. K. and D. H. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 409-862-1433; Fax: 409-862-4929; E-mail: fschroeder@cvm.tamu.edu.

Published, JBC Papers in Press, May 31, 2000, DOI 10.1074/jbc.M000431200

    ABBREVIATIONS

The abbreviations used are: SCP-2, sterol carrier protein-2; pro-SCP-2, 15-kDa pro-sterol carrier protein-2; 13-kDa SCP-2, sterol carrier protein-2; SCP-x, 58-kDa sterol carrier protein-x; cis-parinaric acid, 9Z,11E,13E,15Z-octadecatetraenoic acid; trans-parinaric acid, 9E,11E,13E,15E-octadecatetraenoic acid; dehydroergosterol, Delta 5,7,9(11),22ergostatetraen-3 beta -ol; NBD-cholesterol, 22-(N-7-nitrobenz-2-oxa-1,3-diazo-4-yl)-amino-23,24-bisnor-5-cholen-3beta -ol)); FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption-induced/time-of-flight.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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