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Originally published In Press as doi:10.1074/mcp.M600143-MCP200 on July 18, 2006.
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Molecular & Cellular Proteomics 5:1647-1657, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Differential Expression of Serum Clusterin Isoforms in Colorectal Cancer*

Ana M. Rodríguez-Piñeiro{ddagger},§, María Páez de la Cadena{ddagger}, Ángel López-Saco and Francisco J. Rodríguez-Berrocal{ddagger},||

From the {ddagger} Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Campus Universitario, 36310 Vigo, Spain and Servicio de Cirugía, Complejo Hospitalario Universitario de Vigo, 36104 Vigo, Spain


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.

Clusterin (CLU)1 (otherwise called apolipoprotein J, sulfated glycoprotein-2, or testosterone-repressed prostate message-2 among others) is a highly conserved, secreted heterodimeric glycoprotein. CLU has been implicated in diverse physiological processes, including lipid transportation (1), complement inhibition (1), tissue remodeling (2), membrane recycling (3), and clearance of cellular debris (4). It has also been related to cell-cell and cell-substratum interactions as a potent cell aggregation and adhesion molecule (5). CLU is induced in many severe physiological disturbances including neurodegenerative pathologies (6). In cancer, CLU up-regulation has been described in renal cell carcinoma (7), breast carcinoma (8), ovarian cancer (9), anaplastic large cell lymphomas (10), desmoplastic melanoma (11), transitional cell carcinoma of the bladder (12), pancreatic cancer (13), and serous carcinoma and hepatocellular carcinoma (14). However, a number of tumor processes where CLU is down-regulated have also been described such as esophageal squamous cell carcinoma (15), testicular germ cell tumors (16), and prostate cancer (17).

Regarding CLU and human colorectal cancer (CRC), Chen et al. (18) reported that this protein is elevated in early intestinal lesions, benign polyps, adenocarcinomas, and normal epithelia adjacent to tumors. Recently an overexpression of the secreted CLU in highly aggressive colon tumors and metastatic nodes has been reported (19).

Despite the efforts to understand CLU function, it has remained elusive for years. CLU has been described to participate in distinct and usually opposing functions, and it has been proposed that most likely it is a member of the large family of molecules that orchestrate the immediate cellular response to any type of insult and regulate the balance between cellular growth/arrest and survival/death (20).

One of the reasons for the disparity of functions might be the existence of multiple isoforms. CLU is encoded by a single copy gene of nine exons, spanning over 16 kb and located at chromosome 8p21-p12. The primary translation product is a polypeptide of 449 amino acids (21), containing a 22-mer signal peptide, generating a non-glycosylated holoprotein (precursor form, pCLU) with a predicted molecular mass of 60 kDa. Maturation of this polypeptide includes bridging by five disulfide bonds (22), conversion to a high mannose endoplasmic reticulum-associated form (23), extensive additional N-linked glycosylation (24), and proteolytic cleavage in the trans-Golgi or post-Golgi compartments. Other post-translational modifications, such as sulfation, iodination, and mannose-6-phosphorylation, have been reported (25). The mature product is a secreted protein of 70–80 kDa (sCLU) that under reducing conditions yields two subunits ({alpha} and ß chains) of around 40 kDa (26). sCLU acts as a molecular chaperone, scavenging denatured proteins outside the cells following specific stress-induced injury (27). Interestingly the overexpression of sCLU in human cancer cells caused drug resistance and protection against certain cytotoxic agents that induce apoptosis (28, 29). It has just recently been demonstrated that this antiapoptotic effect of CLU is mediated by interfering with Bax oligomerization in mitochondria, impeding the release of cytochrome c and the activation of caspases (30). Conversely a precursor nuclear CLU (nCLU) is produced by an alternative splicing of the CLU gene, resulting in a protein of 49 kDa that is present in a dormant state in the cytosol of human cells. After exposure to proapoptotic signals, this inactive form of the nCLU is post-translationally modified by an unknown mechanism, producing an active 55-kDa protein that accumulates in the nucleus and causes cell death (31).

CLU has been regarded as a potential indicator for diagnosis of human CRC (18), and a possible prognostic and predictive role as a marker for colon carcinoma aggressiveness has been suggested (19). In those recent works, authors proved a correlation between the presence of a particular intracellular isoform and tumor progression, suggesting that the controversy on CLU function in tumors may be related to the pattern shift of its isoform production.

Because CLU is a secreted protein, it has been found in extracellular fluids, but to date there are not many studies regarding CLU in serum. Lakins et al. (32) studied serum CLU in rat; Watanabe et al. (33) detected overexpression of serum CLU in preeclamptic women; Doustjalali et al. (34) described the behavior of serum CLU in breast cancer. All those studies revealed a plethora of circulating CLU isoforms.

To our knowledge, the present work is the first in which serum CLU and its isoforms have been studied in CRC patients. To know the normal pattern of extracellular CLU, we applied classical and new proteomic techniques to separate and analyze its isoforms in serum from donors. Then to find out whether serum CLU was altered due to the CRC disease, we compared the isoforms in serum of CRC patients with those of healthy donors, finding several that altered their level of expression in relation to the cancer process. Finally the potential utility of serum CLU and its isoforms as CRC markers was tested by immunoquantification.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Collection and Preparation—
Preoperative blood was obtained by venipuncture from 10 patients who were operated on for CRC at Complejo Hospitalario Universitario de Vigo (Vigo, Spain). Blood samples of the control group (10 individuals: six males and four females), provided by the Galician Transfusion Center, were from healthy, habitual, and controlled blood donors. All the donors included were over 50 years old to match the age range of the patients. Both for donors and patients, drawn blood was allowed to coagulate at room temperature for 15 min and centrifuged at 2,000 x g for 15 min. Once obtained, sera were stored at –85 °C. All procedures involving human samples were performed according to the clinical ethical practices of the Spanish Government and followed the tenets of the Helsinki Declaration. Informed consent was obtained from each subject’s guardian, and anonymity was warranted tracing the patients through their clinical history number.

Pathological specimens from CRC patients were processed for regular pathological and histological examination. The stage of tumors was established according to the TNM classification (35). The degree of histological differentiation was described by the pathologist, and tumor location was determined from the surgeon report (Table I).


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  		TABLE I Clinical and anatomopathological characteristics of the cases analyzed

 
Concanavalin A-Sepharose Affinity Chromatography—
The chromatographic method was performed as published elsewhere (36). Briefly 1 ml of filtered serum was applied to a concanavalin A (Con A)-Sepharose (Sigma) column (0.8 x 7-cm Econo column, Bio-Rad) equilibrated in 10 mM sodium/disodium phosphate buffer, pH 6.0. Flow was stated at 0.3 ml/min. After applying the serum, the column was first washed with 30 ml of equilibrating buffer, releasing a fraction enriched in non-glycosylated proteins and O-glycoproteins (FI). Then N-glycoproteins (FII) were selectively eluted with 0.5 M methyl {alpha}-D-mannopyranoside. OD at 280 nm was measured along the chromatographic process using a spectrophotometer (UVIKON 930, Kontron Instruments). Protein concentration was measured according to Lowry et al. (37). Chromatographic fractions were dialyzed against milliQ water at 4 °C overnight, frozen, lyophilized, and then stored at –85 °C until used.

One-dimensional Gel Electrophoresis—
For denaturing electrophoresis (SDS-PAGE), 20 µg of protein from total serum and from the chromatographic fractions were resuspended in sample buffer and resolved in 12% (v/v) polyacrylamide denaturing minigels according to Laemmli (38). Native PAGE was performed likewise, only substituting SDS for an equivalent volume of milliQ water and handling the samples at 4 °C. Ammoniacal silver staining was performed following the protocol from Heukeshoven and Dernick (39).

Deglycosylation with Peptide-N-glycosidase F (PNGase F)—
Deglycosylation of CLU was performed with the enzyme PNGase F (peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase, EC 3.5.1.52) recombinant (Roche Diagnostics). Glycans were released following the protocol described by Ausubel et al. (40). Transferrin was used as a positive control of the enzyme activity.

Two-dimensional Gel Electrophoresis—
Lyophilized fractions were solubilized by suspension in lysis buffer (7 M urea, 2 M thiourea, and 4% (w/v) CHAPS) for 3 h at 30 °C with vigorous shaking in an orbital incubator (Gallenkamp, B. Braun Biotech). This mixture was assayed for protein concentration according to Bradford (41) with modifications (42), then aliquoted in appropriate volumes, and stored at –20 °C. For IEF, 150 µg (large analytical gels) or 200–500 µg (preparative minigels) of sample were mixed with rehydration buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.3% (w/v) DTT, and 0.5% (v/v) Bio-Lytes 3/10 up to 350 or 150 µl, respectively. This mixture was used to rehydrate 17- or 7-cm, pH 4–7, linear ReadyStripTM IPG Strips (4% T, 3% C) (Bio-Rad) for 12 h at 20 °C with a constant voltage (50 V) applied across the gel strips, which were placed in the Protean IEF cell (Bio-Rad) focusing tray. The rehydrated large gels were electrophoresed at 250 V for 15 min, subjected to a linear voltage ramp from 250 to 10,000 V for 5 h, and then focused to 60,000 V-h. Minigels were electrophoresed at 250 V for 15 min, up to 4,000 V for 3 h, and finally to 28,000 V-h. The IPG strips were then placed in SDS-PAGE equilibration buffer containing 6 M urea, 50 mM Tris, pH 8.8, 2% (w/v) SDS, and 30% (v/v) glycerol with 1% (w/v) DTT for 20 min. The procedure was repeated with SDS-PAGE equilibration buffer with 2.5% (w/v) iodoacetamide for an additional 20 min (43). After equilibration, the IPG gel was transferred onto a 9–16% gradient polyacrylamide gel for large IPGs or a 12% minigel for small strips (30% T, 2.6% C). SDS-PAGE was performed in a Protean II xi cell or a Mini-Protean II cell (Bio-Rad). Finally gels were either silver-stained or transferred to PVDF membranes. For MS identification, 1.5 mg of protein were resolved following the protocol used for large analytical gels, and 2-D gels were stained with Coomassie Brilliant Blue as described by Rosenfeld et al. (44).

Blot and Immunodetection—
Proteins were bound to PVDF membranes (Immobilon-P, Millipore) either by Western blot using a Mini-Protean II cell or by slot blot using the Bio-Dot SF apparatus (Bio-Rad). For slot blotting, samples were diluted 1:1,000 for total serum and 1:10 for fractions, both in PBS. Blanks were made with PBS. The linearity of the assay was calculated for dilutions of both total serum and the chromatographic fractions, obtaining correlation coefficients greater than 0.995 for all the types of samples.

After blotting, membranes were blocked with 5% (w/v) nonfat milk in PBS, pH 7.4. Immunodetection of CLU was performed either with 1 µg/ml (Western blot) or 5 µg/ml (slot blot) mouse anti-CLU {alpha} chain (human), clone 41-D, monoclonal antibody (Upstate Biotechnology) or 1 µg/ml rabbit polyclonal anti-CLU-{alpha}/ß (H-330) (Santa Cruz Biotechnology). Membranes were washed thrice with PBS and incubated with 1:2,000 secondary antibody (anti-mouse and anti-rabbit, respectively, IgG alkaline phosphatase conjugate) (Bio-Rad) after Western blot or 1:1,000 secondary antibody after slot blot. Bands were visualized by adding the chromogenic substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics).

Computer Analysis of Electrophoretic Patterns—
Gels and blots were digitized with a calibrated densitometer (GS-800, Bio-Rad). 1-D images were analyzed with the Quantity One 4.4.1 software (Bio-Rad). Molecular weights for proteins resolved by PAGE were estimated with the appropriate protein standards for each technique. Regarding 2-D PAGE, protein patterns were compared using the PDQuest 7.1.1 software (Bio-Rad). The intensity levels of the spots were normalized by expressing the intensity of each spot in a gel as a proportion of the total protein intensity detected for the entire gel (relative volume) (45).

Statistical Methods—
The statistical significance of the relative levels of CLU was assessed with the non-parametric Mann-Whitney U test. Analyses were done using the SPSS software package (release 11.5). p values ≤0.05 were considered statistically significant.

Mass Spectrometric Protein Identification—
Altered spots from 2-D gels belonging to the CLU area were identified by mass spectrometric methods as described elsewhere (36). Briefly spots were excised from Coomassie-stained gels and destained with 50 mM ammonium bicarbonate and 50% (v/v) ACN. Then gel pieces were dried and digested with 10 µg/ml trypsin in 25 mM ammonium bicarbonate at 37 °C overnight. Peptides were eluted with 5% (v/v) TFA and 75% (v/v) ACN. Finally samples were mixed with an {alpha}-cyano-4-hydroxycinnamic acid matrix and analyzed on a M@LDI-HTTM (Micromass). Data processing was performed with MassLynx (Micromass), and peptide fingerprints were searched against non-redundant National Center for Biotechnology Information (nrNCBI) and Swiss-Prot databases by MASCOT Daemon search engine (Matrix Science Ltd., London, UK).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Concanavalin A Chromatography of Serum from Donors and Patients—
We performed chromatography through the lectin Con A to separate human serum, both from donors and patients, into two fractions. This lectin binds proteins with complex- and high mannose-type oligosaccharide chains, thus obtaining a flow-through fraction including albumin and other serum proteins mainly non-glycosylated and O-glycosylated (hereafter called FI) and a second eluate enriched in N-glycoproteins (hereafter called FII) (46). For this work, we processed sera from 10 donors and 10 CRC patients; clinical and pathological features of each CRC case are summarized in Table I. In Fig. 1, we show a representative chromatographic profile for each group and the recovery of protein in each fraction regarding the whole serum.


Figure 1
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  		FIG. 1. Representative chromatographic profiles of serum from donors and CRC patients. The amount (in mg) of protein processed through the column matrix and recovered in each fraction, calculated for the 10 donors and the 10 patients included in this work, is shown.

 
Immunodetection of CLU in 1-D Blots—
To detect the serum isoforms of CLU in donors and patients, proteins from total serum and chromatographic fractions FI and FII were submitted to native PAGE, Western blot, and immunodetection with anti-CLU monoclonal antibody (41-D). Native analysis (Fig. 2) revealed a smeared band ranging from ~70 to 85 kDa in the total serum (S) and in the chromatographic fraction FII both for donors and CRC patients. This mass range fits with the expected molecular mass for the sCLU, corresponding to the glycosylated heterodimer. We also found immunoreaction at higher molecular masses, especially in the FII where we could distinguish neat bands of 92 and ~110 kDa. In addition, bands of ~140, ~150, and ~160 kDa were noticed both in S and FII. The most outstanding difference between donors and CRC patients was observed in the chromatographic fraction FI. In this case, we could observe in patients an intense immunoreactive band (~85 kDa), which was almost undetectable in donor samples, that showed three faint forms reacting with the antibody (~60, 68, and 78 kDa).


Figure 2
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  		FIG. 2. Immunodetection of CLU by the monoclonal antibody against human {alpha} chain after native PAGE of 20 µg of protein from representative samples. MW, molecular mass markers; dFI, donor fraction I; dS, donor serum; dFII, donor fraction II; pFII, patient fraction II; pS, patient serum; pFI, patient fraction I. Plots show the reflective density; each peak is labeled with its molecular mass (kDa).

 
Under reducing conditions (SDS-PAGE) (Fig. 3) we found in serum samples a characteristic set of three bands (~80, 85, and 90 kDa) that was easily seen when the samples were resolved in a 10% gel (Fig. 3B) and six different bands of about 40 kDa (coincident with the mass of each of the two subunits, {alpha} and ß, described for CLU) for which the distribution of mass ranges was slightly different for donors and patients (Fig. 3A). FII showed two bands of ~75 and ~80 kDa (Fig. 3B) and five bands of about 40 kDa (Fig. 3A) with no apparent differences between donor and patient fractions. On the other hand, FI did not present bands within high mass ranges for either donors or patients. Noticeably the major change was found again in FI from patients where the 85-kDa band previously found in native conditions was now reduced to a subunit of 40–41 kDa that was absent from the FI of donors. Nonetheless in the latter, two faint bands (30 and 36 kDa, corresponding to the 60, 68, and 78-kDa bands seen in native-PAGE) were hardly visible.


Figure 3
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  		FIG. 3. Immunodetection of CLU by the monoclonal antibody against human {alpha} chain after 12% (A) or 10% polyacrylamide SDS-PAGE (B) of 20 µg of protein from representative samples. MW, molecular mass markers; dFI, donor fraction I; dS, donor serum; dFII, donor fraction II; pFII, patient fraction II; pS, patient serum; pFI, patient fraction I. Plots show the reflective density; each peak is labeled with its molecular mass (kDa).

 
These results were repeatedly found in all samples analyzed. Moreover the same band pattern for S, FI, and FII was detected after immunoreaction with the H-330 polyclonal antibody against {alpha} and ß chains (data not shown).

Deglycosylation with PNGase F of the CLU Isoform Found in FI from Patients—
To determine whether the 40-kDa immunoreactive band observed in FI of CRC patient sera is glycosylated, we used the enzyme PNGase F that cleaves all types of asparagine-bound N-glycans provided that the amino group and the carboxyl group are present in a peptide linkage and the oligosaccharide has the minimum length of the chitobiose core unit. After treatment with PNGase F, we found that the 40-kDa band was reduced to a 27.8-kDa band (blot not shown), corresponding to the polypeptidic backbone of the protein, indicating that the aberrant isoform is N-glycosylated. Transferrin (resolved as 85.3 kDa) was used as a positive control for the enzyme activity; it was cleaved to 79.9 kDa (77 kDa predicted from its amino acid chain).

Immunodetection of CLU in 2-D Blots—
To get a better separation of CLU isoforms, FI and FII fractions from controls and patients were submitted to 2-DE, and then immunodetection with the monoclonal anti-CLU antibody was performed (Fig. 4). In the FI fraction of donors, CLU was slightly immunodetected around 35 kDa. However, this technique clearly reveals that the band observed in the FI fraction of patients (see Figs. 2 and 3A) contained five isoforms with a mass of ~40 kDa. On the other hand, in FII fractions, besides the expected 40-kDa isoforms, we detected immunoreaction in a cluster of 75–80 kDa, corresponding to the smear already found in denaturing 1-D gels (see Fig. 3).


Figure 4
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  		FIG. 4. Immunodetection of CLU in denaturing 2-D blots. Upper left, FI from a donor where a weak signal was detected for at least three ~35-kDa isoforms. Upper right, FI from a patient displaying the ~40-kDa CLU as five isoforms in regard to their pI. Bottom left and right, FII from a donor and from a patient, respectively, clearly displaying the 35–45- and 75–80-kDa CLU forms. Insets show a tridimensional view of the isoforms displayed by the PDQuest software.

 
CLU Analysis by 2-D PAGE—
The Western blot technique is not completely reliable for quantification because the transfer process could introduce artifacts altering the ratio between spots. Moreover the different CLU isoforms both in donors and patients were displayed in a crowded area after separation. Thus, to accomplish a semiquantitative comparison of CLU isoforms between donors and patients, we performed 2-D PAGE of FII fractions using large gels that spatially expanded the pI range, and gels were stained with silver nitrate (Fig. 5).


Figure 5
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  		FIG. 5. Representative silver-stained 2-D gels from donor FII (left) and CRC patient FII (right). Rectangles contain the area where the 40-kDa CLU isoforms are displayed after 2-D separation. Superimposed to the maps we show the three-dimensional views of the CLU area where the spots listed in Table II are numbered.

 
We studied the isoforms described in the 40-kDa protein cluster available in the SWISS-2DPAGE plasma reference map (www.expasy.org/cgi-bin/ch2d-compute-map?PLASMA_HUMAN,P10909) locating 16 spots (Fig. 5, boxes) in FII maps from both donors and patients. We could not locate the cluster of 75–80 kDa in large silver-stained gels because we did not have enough landmarks in 2-D Western blots (Fig. 4) that could locate CLU in relation to other proteins displayed in the fraction.

Table II shows the pI and molecular mass of the located CLU isoforms, their amount measured in donors and patients by means of the spot relative volume, and the statistically significant alterations. Most of these spots were decreased in patients, and only spot number 1215 was increased by 2-fold in patients. Six of the 16 isoforms were absent or not detected in patient maps, and therefore the variation could not be calculated. All those altered spots sent for MS identification were confirmed to be CLU.


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  		TABLE II Differential expression of serum CLU isoforms found in 2-D maps of control and CRC patient FII fractions

 
Immunoquantification of CLU by Slot Blot—
To test the potential utility of serum CLU as a tumor CRC marker, we measured the amount of the protein in 10 donors and 10 patients. Because there was no ELISA commercially available, we used a slot blot technique followed by immunodetection of the protein with the monoclonal antibody. Quantification was done by densitometry using the Quantity One 4.4.1 software.

The relative amount of CLU in total serum, FI, and FII was measured as the optical density in reference to the protein content (OD/mg). The results of the quantification are shown in Fig. 6 as the OD/mg values found for each of the 10 donors and the 10 CRC patients tested. In total serum, the amount of CLU was significantly overexpressed in patients (56.92 ± 6.90) with regard to donors (42.27 ± 6.60; p = 0.001). Similarly we detected a significant increase of the FI CLU amount in patients (4.79 ± 2.65 versus 0.60 ± 2.09; p = 0.013). FII CLU showed a decreasing trend (109.68 ± 25.10 versus 130.47 ± 25.94) as inferred from the 2-D PAGE results, although this tendency was not significant (p = 0.096).


Figure 6
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  		FIG. 6. Amount of CLU protein in total serum and FI and FII chromatographic fractions measured as the optical density in reference to the protein content (OD/mg). Circles represent the values for the 10 donors and the 10 CRC patients tested; the horizontal line shows the median of the distribution. Statistical assessment by the Mann-Whitney U test demonstrated significant differences for total serum CLU (p = 0.001) and FI CLU (p = 0.013).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The study presented here is based on the analysis of serum CLU and its different isoforms in relation to CRC. In recent years, many works have focused their attention on the relationship between CLU and cancer (7–19), and in particular, studies in CRC tumors have demonstrated an oncogenic role of CLU in carcinogenesis and overexpression related to tumor progression (9, 18, 19). Consequently and because it is well known that CLU is a protein that is secreted to body fluids (26), it has been suggested that quantification of CLU in serum may have a potential utility for diagnosis and prognosis of human CRC (18, 19). However, to our knowledge there are no published studies about CLU levels in serum of CRC patients until now.

When approaching this work we kept in mind the complexity of the protein because CLU can appear as multiple isoforms not only in tissues (19, 31, 48) but also in serum. This fact is reflected, for instance, in the human serum proteome map where at least 16 isoforms of CLU are annotated (www.expasy.org/cgi-bin/ch2d-compute-map?PLAS-MA_HUMAN,P10909). It is obvious that, because CLU is expressed in a wide range of tissues, serum CLU would represent a collection of different isoforms that could appear in serum depending on the physiological status or in relation to a disease. Regarding cancer, some authors have concluded that alternative isoforms of CLU could participate in processes that may have opposite effects, such as apoptotic and antiapoptotic roles. One relevant study was published by Pucci et al. (19) suggesting that a different CLU function in CRC tumors may be related to a shift in the pattern of its isoforms production. They described an overexpression of the sCLU, which occurs in the cytoplasm of highly infiltrating tumors and metastatic nodes. The increased level of the secreted form and the disappearance of the nuclear unglycosylated form are directly connected to increased cell survival, aggressiveness, and enhanced metastatic potential. Regarding the nCLU, Chen et al. (49) described an increase both of the CLU RNA and protein levels when apoptosis was induced in human colon cancer lines, localizing the protein at the nuclei and supporting the proapoptotic role of nCLU. Moreover they found that this effect was dependent on p21 but not on p53 expression. On the other hand, Caccamo et al. (48) have investigated the possible connection between CLU and apoptosis, suggesting that changes in the steady-state balance between CLU isoforms could differently affect cell growth, possibly priming cells to death or allowing cell survival instead. In both publications, the authors suggested that the different behavior of particular CLU isoforms is the most likely explanation for the contradictory data on CLU function.

Considering this background, in the first part of our work we studied the isoforms of CLU in human serum both in healthy donors and in CRC patients. We took advantage of the development of proteomic techniques and the existence of monoclonal anti-CLU antibodies to separate, identify, and quantify the different isoforms.

In a previous work, we reported the use of Con A-Sepharose chromatography combined with 2-D PAGE as a consistent, reproducible, and reliable method for comparative proteomic studies of serum (36). Con A is a plant lectin that interacts with high mannose-type and hybrid-type sugar chains of N-glycoproteins (50). The main advantage of using this chromatography in CLU studies prior to other techniques, especially electrophoresis, is that we can get a serum fraction enriched in the protein of interest (because CLU is an N-glycosylated protein (24, 51)), avoiding the interference of albumin, which is an unglycosylated protein.

In the present study, the chromatographic process allowed the separation of serum samples into two fractions: FI, enriched in non-glycosylated and O-glycosylated proteins, and FII, enriched in N-glycoproteins. After the chromatographic separation, total serum, FI, and FII were resolved by 1-DE, blotted, and immunodetected with anti-CLU antibodies. Under native conditions, in total serum and FII we observed the expected heterodimer (~70–85 kDa), which corresponds to the amino acidic part of the protein that is fully or partially glycosylated (24). In addition we found high molecular weight forms in total serum that could represent aggregates of CLU together with apolipoprotein A-I, paraoxonase, and/or cholesteryl:ester transfer protein because these molecules are co-localized in plasma in high density lipoprotein particles (26), aggregates with components of the soluble terminal complement complex, or even dimers of the holoprotein or the mature heterodimer.

When a denaturing treatment was applied before running 1-DE, we still detected bands in the range of 70–85 kDa in serum and in FII that could correspond to the fully or partially glycosylated holoprotein. Major immunoreactions in those samples were due to ~40-kDa bands that corresponded to the CLU {alpha} subunit. Although a number of bands could be distinguished, no significant alteration in their quantity was found between donors and patients.

Both under native and denaturing conditions, an unexpected CLU form was revealed in FI from patients (~85 and 40 kDa, respectively). This form eluted in the unbounded fraction of serum through Con A chromatography, so it was expected to be a non- or O-glycosylated form. Nevertheless because the band was measured as a 40-kDa form and considering that the predicted mass for the amino acidic sequence of either {alpha}-CLU or ß-CLU was not more than 30 kDa, we could not rule out the possibility of glycosylation of this form. On the other hand, thus far no O-glycosylation of CLU has been detected in glycosylation studies (24, 51). Therefore, the 40-kDa chain found in FI of patients was subjected to deglycosylation with PNGase F to prove whether it was N-glycosylated because this enzyme releases asparagine-linked (N-linked) oligosaccharides from glycoproteins (52). After deglycosylation, the immunoreacting band of 40 kDa shifted to ~28 kDa, a mass that corresponds to the amino acidic sequence of a CLU chain. Therefore, we concluded that the patient FI CLU band was glycosylated by N-linkage. The reason why it eluted in FI, instead of being retained by the lectin, will be further studied, although we hypothesize that an abnormal N-glycosylation of some specific isoforms could occur in cancer patients. In fact, it is well known that in CRC patients there is an increase of 53% for bound sialic acid in serum (53). It was also established that the synthesis of N-glycans with poly-N-acetyllactosamine side chains is associated with tumor cells showing high metastatic capacity compared with those with low metastatic potential (54). It is also a fact that an aberrant glycosylation decreases the affinity of an oligosaccharide to the Con A (46, 55–57). Hence should it be due to an increased sialylation or to a change in structure, the fact is that the CLU isoform eluted in FI does not bind to the Con A matrix and specifically appears in relation to the CRC disease.

We have analyzed FI and FII samples by 2-D PAGE followed by immunodetection of CLU. In relation to FI, an extremely weak signal was detected in fractions from donors, whereas in patients the intense 40-kDa band observed in denaturing 1-D gels was revealed in 2-D gels as three intense and two fainter spots; thus there are at least five different isoforms of CLU in the CRC patient group. As expected, bearing in mind the results of 1-D SDS-PAGE, the pattern resolved in 2-D maps of FII fractions was further complicated. Interestingly we found high molecular mass isoforms (75–80 kDa) corresponding to the bands observed in FII by 1-DE. However, this result is surprising if one considers the strong denaturing conditions of 2-D PAGE. We hypothesized that they are most probably the mature holoprotein, not proteolytically cleaved. Both for control and patient samples, we found the expected 40-kDa isoforms in FII. We observed a different pattern when comparing profiles of donors and patients, but still we consider that the blotting process could introduce variability and alter their relative amounts making it inadequate to quantify the difference by this technique.

Therefore, to better compare the CLU FII isoforms, we displayed them by analytical 2-D PAGE, expanding the 4–7 pI range in larger gels and visualizing the spots after silver staining. CLU has been detected previously in 2-D maps of the serum proteome with the appearance of a train of spots (33, 34). In general, it is believed that this type of spot "constellation" is caused by a variability in sialylation of the different protein glycoforms, and their differential expression could make them useful as markers of disease (58). Hence samples from donors and patients were studied, comparing the relative volume (resembling quantity) of the spots appearing in the CLU area. We were able to visualize differences that could not be detected by 1-D Western blot: a 2-fold increase was observed in one of the isoforms identified, whereas the relative volume of the rest of the CLU isoforms in FII was decreased in patient maps compared with donor maps or even absent in patients.

The conclusion of our proteomic studies is that specific serum isoforms of CLU are different in amount and/or structure in CRC patients. In summary, we found five FI and one FII CLU isoforms that are increased in serum of CRC patients and 15 FII isoforms that are decreased/absent.

After the proteomic analyses the question that remained was whether the detection and quantification of CLU in serum of CRC patients could reflect the relationship of the protein with the disease. Until now, not many studies regarding the CLU content in serum have been published. This might be due to the lack of a commercial kit to test the amount of CLU in blood. Nevertheless some immunoassays have been developed by different groups (33, 34, 59, 60). For instance, Morrissey et al. (59) developed and validated an antigen capture assay in an attempt to determine whether CLU levels in human serum could be of diagnostic or prognostic utility regarding prostate cancer. The average level of the protein measured thereby was 101 ± 42 µg/ml in good agreement with a variety of other assays where CLU level had been reported from 35–105 µg/ml (61) to 111 ± 50 µg/ml (62). However, the lack of purified human serum protein to use as standard is the reason that many studies report CLU levels as -fold variation or in OD units, making it difficult to compare results from different laboratories. As an example, Trougakos et al. (60) have set up a sandwich assay for serum CLU, finding an increased OD for the measurement of CLU in type II diabetes, coronary heart disease, and myocardial infarction.

In our study, to measure the amount of serum CLU we used a very simple method. Proteins were slot-blotted on PVDF membranes and then immunodetected with a monoclonal antibody. The results were then comparable to those of an immunoassay because there was no previous electrophoretic separation. We quantified CLU in total serum and in FI and FII chromatographic fractions, comparing the results obtained for 10 donors with those for 10 CRC patients. Regarding total serum, the amount of CLU was significantly increased in patients. Because the distribution of serum CLU levels did not show much overlap between donors and patients, it could be an interesting target for extensive diagnostic studies. In FII, despite the differences detected in 2-DE studies, almost no variation was observed in the CLU level, although this is not surprising because at least 16 isoforms were measured at once. On the other hand, the most outstanding result of the immunoquantification of CLU appeared in patient FI where the amount of CLU was significantly increased an average of 8-fold when compared with FI from healthy donors. This perfectly agrees with the previous results obtained by Western blot where a 40-kDa form appeared overexpressed in FI of patients. Despite the significant differences found between cohorts, the individual FI CLU values measured by immunoquantification display an overlapped area that would affect the sensitivity and specificity of this form as a tumoral marker for CRC. This is probably due to the detection not only of the 40-kDa band (in its native form) specifically found in patients but of the ~30-kDa bands (in their native forms) detected in donors (see Figs. 2 and 3A). Therefore, it would be more useful to separate the isoform of interest and specifically quantitate it alone.

The FI isoform of CLU found to be increased in serum of CRC patients could correspond to that recently described by Pucci et al. (19). They found that in high grade carcinomas with metastatic nodes there is a cytoplasmic overexpression of a highly glycosylated form of CLU. They also confirmed in vitro that this form is released to the extracellular space. The CLU form found in the FI of patients in our study is also glycosylated (probably in an aberrant manner) and corresponds in size to that described by Pucci et al. (19).

Previous examples of the utility of specific isoforms of a protein have been reported. For instance, Stulík et al. (63) found that calcyclin (a Ca2+-binding protein) exhibits different isoforms (I–IV) whose abundance is significantly varied in colon carcinomas, suggesting that calcyclin expression pattern could indicate the progression of malignant lesions toward neoplasms. Another example was shown by Charrier et al. (47) who demonstrated that the prostate-specific antigen presents standard and low molecular weight isoforms; the latter are increased in patients with prostate cancer compared with those with benign hyperplasia. Similarly we think that some isoforms of CLU could be more informative in relation to the CRC pathology than the total serum CLU. Because there is a current need of new circulating markers for CRC, the potential utility of the CLU isoforms should be explored in the future.


   ACKNOWLEDGMENTS
 
We thank Gloria Otero for technical assistance. We also thank "Centro de Transfusión de Galicia" and "Complejo Hospitalario Universitario de Vigo" for providing samples, especially Carmen Díaz-Rois for blood collection.


  FOOTNOTES
 
Received, April 18, 2006, and in revised form, December 12, 2006.

Published, MCP Papers in Press, July 18, 2006, DOI 10.1074/mcp.M600143-MCP200

1 The abbreviations used are: CLU, clusterin; CRC, colorectal cancer; sCLU, secreted clusterin; nCLU, nuclear clusterin; TNM, tumor, node, metastases; Con A, concanavalin A; PNGase F, peptide-N-glycosidase F; 2-D, two-dimensional; 2-DE, two-dimensional gel electrophoresis; 1-D, one-dimensional; 1-DE, one-dimensional gel electrophoresis; S, serum. Back

* This work was supported in part by "Xunta de Galicia" Grants PGIDIT02BTF30103PR and PGIDIT03PXI30103IF. Back

§ Supported by a predoctoral fellowship from the Ministerio de Educación y Cultura (Spain). Back

|| To whom correspondence should be addressed. Tel.: 34-986-812571; Fax: 34-986-812556; E-mail: berrocal{at}uvigo.es


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 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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