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Clinical Cancer Research Vol. 12, 6367-6372, November 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Identification of the Decay-Accelerating Factor CD55 as a Peanut Agglutinin–Binding Protein and Its Alteration in Non–Small Cell Lung Cancers

Mitsunori Higuchi1, Yuichi Endo2, Hiroyuki Suzuki1, Fumihiko Osuka1, Yutaka Shio1, Koichi Fujiu1, Ryuzo Kanno1, Akio Oishi3, Teizo Fujita2 and Mitsukazu Gotoh1

Authors' Affiliations: Departments of 1 Surgery I and 2 Immunology, Fukushima Medical University School of Medicine; and 3 Fukushima Red Cross Hospital, Fukushima, Japan

Requests for reprints: Mitsukazu Gotoh, Department of Surgery I, Fukushima Medical University School of Medicine, 1-Hikariga-oka, Fukushima 960-1295, Japan. Phone: 81-24-547-1254; Fax: 81-24-548-2735; E-mail: mgotoh{at}fmu.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Peanut agglutinin (PNA) recognizes tumor-associated carbohydrates. In this study, we aimed to identify the core protein harboring PNA-binding sugars in the human lung and to explore the relationship with the pathology of primary non–small cell lung cancers (NSCLC).

Experimental Design: PNA lectin blotting was used to detect PNA-binding proteins in the microsomal fraction of lung tissue from 24 patients with NSCLC. The 55- to 65-kDa core peptide PNA-binding protein was characterized by enzymatic treatment and identified by immunoprecipitation and affinity chromatography. The expression level and increase in size of the 55- to 65-kDa PNA-binding protein/decay-accelerating factor (DAF) were compared between normal and tumor regions of the tumor tissue by Western blotting and quantitative PCR.

Results: The 55- to 65-kDa PNA-binding protein was observed in human lung. This was a glycosylphosphatidylinositol-anchored membrane protein carrying O-linked carbohydrates. This core protein was identified as DAF, one of the complementary regulatory proteins. DAF was enlarged to 65 to 75 kDa in NSCLC tumor lesions due to sialylation in the sugar moiety. At the transcription level, DAF levels were significantly lower in tumor regions, suggesting its down-regulation in NSCLC cells.

Conclusions: DAF was identified as a new PNA-binding protein in the human lung. The down-regulation and heavy sialylation of DAF was associated with pathology in NSCLC, and these alterations make this protein a potential marker for NSCLC.

Carbohydrates on the cell surface play an important role in several metastatic processes by influencing cell-cell and cell-extracellular matrix protein interactions (1). Peanut agglutinin (PNA) is a plant lectin isolated from Arachis hypogaea that preferentially recognizes galactose ß1-3N-acetylgalactosamine linkage in O-linked glycans (2, 3), also called tumor-associated antigen (T-antigen) or Thomsen-Friedenreich antigen (4). The expression levels and localization patterns of PNA-recognizing carbohydrates have been reported to correlate with the aggressiveness of several cancers, including colorectal cancer, breast cancer, and malignant melanoma (1, 3, 5, 6). In lung cancers, however, there are few reports about the relationship between T-antigen expression and clinicopathologic variables (7). We previously reported that the expression of PNA-recognizing carbohydrates was significantly correlated to lymphatic vessel invasion and a high lymph node metastatic rate in lung adenocarcinoma tissue (8). However, the precise mechanism of involvement of PNA-binding sugars in metastasis is unclear and there is no information on the core protein harboring PNA-binding sugars in lung cancers. To date, several PNA-binding proteins or PNA receptors have been identified, including CD8, CD43, CD44, CD45, gp200, polymorphic epithelial mucin, and MGC-24 (9–12).

In the present study, we identified decay-accelerating factor (DAF; CD55) as a new core protein harboring PNA-binding sugars in the lung. DAF is present on all blood elements and most other cell types, especially in high levels on cells that line extravascular compartments. The protein intrinsically functions on cell membranes to protect host cells from autologous complement attack by accelerating the decay of C3 and C5 convertases (13–15). In addition, we report in the current study that DAF was down-regulated and processed to a high molecular weight by sialylation in non–small cell lung cancer (NSCLC) tissue and the pathologic implications are discussed.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue specimens. Tumor lung tissue specimens were obtained from 24 patients with NSCLC who underwent surgery at Fukushima Medical University Hospital (Fukushima, Japan). Specimens included adenocarcinoma, squamous cell carcinoma, and large cell lung carcinoma. Tumors were classified according to the WHO classification of lung tumor (16). All tissue samples were obtained within 30 minutes after surgical resection and stored in liquid nitrogen until further use. Normal, nontumor tissue was obtained from regions sufficiently far away from the cancerous lesion in the same specimen. A signed consent form was obtained from each patient.

PNA lectin blotting. Lung tissue was homogenized in PBS containing protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). Microsome fraction was recovered by differential centrifugation of the homogenate and solubilized by sonication in 0.1 mol/L Tris-HCl (pH 6.8) containing 4% SDS and 20% glycerol (SDS-PAGE sample buffer). Total protein concentration was determined with bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Samples of 20 to 30 µg of protein were subjected to SDS-PAGE on a 7.5% to 10% polyacrylamide gel under nonreducing conditions as described previously (17). Briefly, the blotted membrane was probed with a biotin-conjugated PNA (E.Y. Laboratories, San Mateo, CA) and the color was developed with the Vectastain avidin-biotin complex method kit (Vector Laboratories, Burlingame, CA) and nitroblue tetrazolium (Wako Pure Chemicals, Osaka, Japan).

DAF Western blotting. Western blotting for the microsome DAF fraction was carried out by a protocol similar to the aforementioned PNA lectin blots, except for sequential incubation with anti-DAF antibody (4F11; ref. 18) and then with biotinylated anti-mouse IgG antibody (DAKO, Carpinteria, CA), instead of biotinylated PNA. To quantify the bands in PNA lectin blots and Western blots, the signal intensity of each band was estimated using NIH image software (version 1.56; Wane Rasband; NIH, Bethesda, MD).

Treatment with neuraminidase, endo-{alpha}-N-acetylgalactosaminidase (O-glycanase), and phosphatidylinositol-specific phospholipase C. Selective removal of the terminal sialic acid and all O-linked glycans was achieved by treatment of the microsome fraction with 0.1 unit neuraminidase (Wako Pure Chemicals) alone and with neuraminidase plus 20 milliunits endo-{alpha}-N-acetylgalactosaminidase (Seikagaku Co., Tokyo, Japan), respectively, at 37°C for 16 hours. Treatment of the microsome fraction with phosphatidylinositol-specific phospholipase C (PIPLC; Molecular Probes, Inc., Eugene, OR) was done in PBS containing 0.5 units/mL enzyme at 37°C for 60 minutes.

Affinity chromatography with PNA-agarose column. The microsome fraction from normal tissue was solubilized in TBS containing 1% Triton X-100 and protease inhibitor cocktail (lysis buffer A) at 4°C overnight and subsequently centrifuged at 100,000 x g for 1 hour. The supernatant was loaded onto a PNA-agarose column equilibrated with lysis buffer A. After washing with lysis buffer A, the bound proteins were eluted with buffer containing 0.2 mol/L D-galactose.

Immunoprecipitation with anti-DAF antibody. The microsome fraction from the nontumor region was sonicated in TBS containing 2% Triton X-100, 1 mmol/L EDTA, and protease inhibitor cocktail and then incubated at 4°C overnight. The soluble fraction was recovered by centrifugation at 100,000 x g for 1 hour and mixed with anti-DAF antibody (4F11) at 0°C for 1 hour. Protein G–coupled Sepharose slurry (Sigma Chemical) was added to the mixture and incubated at 0°C for 30 minutes. The bound fraction was recovered with SDS-PAGE sample buffer.

ELISA to quantify soluble DAF. NSCLC tissue was gently homogenized in a Potter-type homogenizer. The 100,000 x g supernatants were incubated in a microtiter plate coated with anti-DAF monoclonal antibody (4F11). Plates were further incubated with biotin-conjugated anti-DAF monoclonal antibody (1C6) and horseradish peroxidase–conjugated avidin (DAKO), and the color was developed using ABTS (Zymed, San Francisco, CA) and H2O2.

Reverse transcription-PCR of DAF, membrane cofactor protein (CD46), and CD59. The expression levels of DAF, membrane cofactor protein (MCP; ref. 19), and CD59 (20) were assessed by reverse transcription-PCR with mRNA isolated from NSCLC tissue using Isogen (Nippon Gene Co. Ltd., Tokyo, Japan).

Real-time PCR was also done to estimate DAF and ß-actin mRNA in an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). The relative gene expression was calculated as a fold induction compared with ß-actin.

Preparation of DAF-overexpressed PC14 cells. Full-length DAF cDNA, which was kindly provided by Dr. D.M. Lublin (Washington University School of Medicine, St. Louis, MO), was cloned in the pcDNA3 plasmid (Invitrogen, Carlsbad, CA). The plasmid was transfected into PC14 cells (21), a human lung carcinoma cell line, by electroporation with Nucleofector (Wako Pure Chemicals). Positive clones were screened by 400 µg/mL neomycin (Geneticin, Life Technologies, Gaithersburg, MD).

DAF immunostaining. Formalin-fixed, paraffin-embedded sections of NSCLC tissue were prepared following standard procedures. Deparaffinized sections were incubated with anti-DAF antibody (4F11) followed by biotinylated rabbit anti-mouse IgG. Color was developed by incubation with 3,3'-diaminobenzidine-H2O2. The sections were counterstained with hematoxylin.

Statistics. Differences in levels of the 55- to 65-kDa PNA-binding protein and DAF between normal and tumor tissues were evaluated by Wilcoxon signed ranks test, and the correlation between both levels was estimated by Pearson's correlation coefficient. The correlation between clinicopathologic feature and the normal/tumor expression ratios of the 55- to 65-kDa PNA-binding protein and DAF was also evaluated with the Student's t test.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
PNA-binding protein (55 to 65 kDa) is present in human lung. PNA lectin blots showed that normal human lung tissue expressed PNA-binding protein with a size of 55 to 65 kDa, termed 55- to 65-kDa PNA-binding protein (Fig. 1A ). The 55- to 65-kDa PNA-binding protein was observed in the microsome fraction of lung tissue homogenate, suggesting its localization on the membranes. Treatment of the lung microsome fraction with endo-{alpha}-N-acetylgalactosaminidase resulted in a complete loss of the 55- to 65-kDa PNA-binding protein (Fig. 1B), indicating that this protein possesses O-linked glycans recognized by PNA. Treatment with PIPLC significantly reduced the amount of 55- to 65-kDa PNA-binding protein in the microsome fraction (Fig. 1B). These results indicate that the core protein is a glycosylphosphatidylinositol-anchored membrane protein with O-linked carbohydrates. The 55- to 65-kDa PNA-binding protein was not detected in peripheral blood or in peripheral hemocytes, indicating that this protein is not a component of peripheral blood that contaminated the lung tissue specimen (data not shown). No 55- to 65-kDa PNA-binding protein was detected in nontumor tissue or tumor tissue from colorectal and stomach cancer (data not shown). As shown in Fig. 1C, the levels of the 55- to 65-kDa PNA-binding protein were significantly lower in NSCLC tumor tissue than in nontumor tissue from the same specimens (P = 0.0002; n = 24). No clear correlation was found between the level of 55- to 65-kDa PNA-binding protein and clinicopathologic variables, such as tumor size, International Union Against Cancer staging, and histology of NSCLC (Table 1 ).


Figure 1
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  		Fig. 1. A, PNA lectin blotting of lung microsome fractions prepared from normal (N) and tumor (T) regions of NSCLC tissue. Two representative cases of patients with adenocarcinoma. B, PNA lectin blotting of the microsome fraction treated with (+) and without (–) PIPLC (left) and endo-{alpha}-N-acetylgalactosaminidase (right). The microsome fraction used was prepared from a normal region of NSCLC tissue. C, levels of 55- to 65-kDa PNA-binding protein in lung tissue. Microsome fractions from normal and tumor regions of 24 patients with NSCLC were subjected to PNA lectin blotting followed by image analysis. The level is represented as an arbitrary unit. Columns, mean; bars, SD. *, P = 0.0002.

 

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  		Table 1. Relationship between clinicopathologic variables and the level of 55- to 65-kDa PNA-binding protein and DAF in 24 patients with NSCLC

 
Identification of DAF as a core protein of the 55- to 65-kDa PNA-binding protein. To identify the core peptide of the 55- to 65-kDa PNA-binding protein, the microsome fraction from lung tissue was solubilized and subjected to affinity chromatography with a PNA-agarose column. The eluate with D-galactose from the column revealed a major band of 55- to 65-kDa by PNA lectin blotting (Fig. 2A ). Based on the structural features of the 55- to 65-kDa PNA-binding protein described above, we suspected DAF (CD55) as a candidate protein. As shown in Fig. 2A, Western blotting with anti-DAF antibody detected a 55- to 65-kDa band in the eluate. Furthermore, anti-DAF antibody pulled down a 55- to 65-kDa band in the immune precipitate from the solubilized microsome fraction (Fig. 2B). These results clearly indicate that one of the core proteins of the 55- to 65-kDa PNA-binding protein is DAF.


Figure 2
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  		Fig. 2. Identification of DAF as the core protein of 55- to 65-kDa PNA-binding protein. A, PNA lectin blotting and Western blotting of bound (B) and unbound (UB) fractions of the solubilized lung microsome to PNA-agarose. B, PNA lectin blotting and Western blotting of immunoprecipitates of the solubilized microsome pulled down with (Ab+) and without (Ab–) anti-DAF antibody.

 
Increased molecular size and down-regulation of DAF in NSCLC tissue. In almost all cases of primary NSCLC, DAF was expressed as a 55- to 65-kDa protein in the nontumor tissue, whereas it was expressed at a larger molecular weight of 65 to 75 kDa in the tumor lesions (Fig. 3A ). Treatment of the microsome fraction with neuraminidase resulted in reduction of the molecular weight of DAF to 55 kDa, both in the nontumor and tumor tissue (Fig. 3B), suggesting the involvement of sialic acid residue. DAF was observed at a larger size of 65 to 75 kDa in a tumor environment using DAF-overexpressed PC14 cells (Fig. 3C). Treatment of the transformants with neuraminidase resulted in a reduction in size to 55 kDa, like native DAF.


Figure 3
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  		Fig. 3. A, Western blotting of DAF in microsome fractions of normal and tumor regions of lung tissue from patients with adenocarcinoma (adeno), squamous cell carcinoma (Sq), and large cell carcinoma (Large). B, reduced molecular mass of DAF by selective removal of sialic acid. The microsome fraction prepared from the normal region of NSCLC tissue was treated with (Neu+) and without (Neu–) neuraminidase. C, Western blotting of DAF in the cell lysate of DAF-overexpressed PC14 cells (Tx+) before (Neu–) and after (Neu+) treatment with neuraminidase. Tx–, native PC14 cells without transfection.

 
In 20 of the 24 cases of NSCLC, the amount of DAF was significantly reduced in tumor tissue compared with nontumor tissue (Fig. 4A ). The soluble form of DAF, which is detached from the membrane or secreted from lung cells as a soluble isoform (14, 22, 23), also decreased in tumor tissue (Fig. 4B). The level of membrane DAF showed no clear correlation with the clinicopathologic variables (Table 1). These results were in close agreement to the PNA lectin blotting findings. As shown in Fig. 4C, a correlation was observed between the level of 55- to 65-kDa PNA-binding protein and DAF (r = 0.582; P < 0.001). Eighteen of the 24 cases that showed a decrease of 55- to 65-kDa PNA-binding protein showed a similar decrease of DAF in tumor tissue. DAF mRNA, assessed by a real-time PCR, was lower in the tumor tissue compared with normal tissue (Fig. 4D), suggesting that DAF is down-regulated in NSCLC tissue at the transcription level. When expression levels of the other complement regulatory proteins were assessed by reverse transcription-PCR, it was found that MCP mRNA was decreased in tumor tissue, but CD59 mRNA in tumor tissue was not significantly different from normal tissue (Fig. 4E).


Figure 4
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  		Fig. 4. A, DAF levels in normal and tumor regions of NSCLC tissue. DAF in the microsome fraction from 24 patients with NSCLC was quantified by Western blotting followed by image analysis. The level is expressed as an arbitrary unit. Columns, mean; bars, SD. *, P = 0.0056. B, levels of soluble DAF in the soluble fractions of NSCLC tissue that were determined by ELISA. **, P = 0.018 (n = 20). C, correlation between the levels of 55- to 65-kDa PNA-binding protein and DAF in the microsome fraction. Open, closed, and gray circles, levels in the normal tissue regions from patients with adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, respectively. Open, closed, and gray squares, corresponding tumor regions. D, DAF mRNA levels estimated by real-time PCR in the normal and tumor regions of NSCLC tissue. DAF mRNA levels are presented as a ratio of DAF mRNA to ß-actin mRNA. Columns, mean; bars, SD. ***, P = 0.003. E, expression of DAF, MCP, and CD59 estimated by reverse transcription-PCR in normal and tumor regions of NSCLC tissue. The levels of DAF, MCP, and CD59 mRNA were evaluated by reverse transcription-PCR. Multiple minor bands were observed for DAF and MCP, which have been identified as splicing variants generated from the DAF and MCP genes, respectively (19, 22). Typical case of a patient with adenocarcinoma.

 
As shown in Fig. 5 , after immunohistochemical staining, DAF was localized to the alveolar epithelial cells in the normal lung, whereas tumor cells were not or very weakly stained. In the tumor tissue, only blood cells and connective tissue were strongly positive.


Figure 5
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  		Fig. 5. Immunostaining of DAF in the normal and tumor regions of NSCLC tissue. Typical result from a patient with adenocarcinoma. DAF staining is observed in alveolar epithelial cells in normal tissue (A), whereas blood cells and connective tissue cells are positive in tumor tissue (B).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that PNA recognized a structure of O-linked carbohydrates on DAF. It is likely that PNA recognizes the limited molecular species of DAF because DAF band detected by Western blot was broader than that seen by PNA lectin blot (Figs. 1-3), although these results might be also explained by the different sensitivities of the two methods. In almost all NSCLC tissues, the expression level of DAF was reduced compared with normal lung tissue. A similar down-regulation was observed in ovarian cancer (24, 25), whereas an up-regulation was reported in other malignancies, such as colorectal cancer and gastric cancer (26–30). From the viewpoint of immunologic surveillance, it is reasonable to suggest that, in a tumor environment, cells are exposed to an augmented complement attack and therefore generate high levels of DAF to protect themselves. The dynamics of DAF in NSCLC tissue require a very different explanation. This down-regulation did not correlate with any specific clinicopathologic variables, suggesting that it is a basal feature of NSCLC. A clue for a possible explanation might lie in the characteristics of sialylated DAF, which should be able to execute its function for longer periods than the regular form because of its high resistance against proteolysis (31). The highly sialylated DAF is retained on the cell surface for a long period, and further biosynthesis of DAF might be suppressed in these tumor cells. Another explanation could lie in the architecture of NSCLC tissue. Unlike normal lung cells, the tumor cells formed a bulky cluster of tissue where the inside was not faced to alveolar cavity (Fig. 5), therefore possibly do not need a high expression of complement regulatory proteins.

Interestingly, a similar down-regulation was observed for MCP, another complement regulatory protein of the regulators of the complement activation family. MCP has a transmembrane protein with a domain structure very similar to DAF. It is likely that DAF and MCP are regulated in a similar manner in NSCLC tissue. However, it is unclear about whether MCP is another 55- to 65-kDa PNA-binding protein, although a small amount of PNA-binding protein was left on the cell membrane after PIPLC treatment (Fig. 1B).

It is of special interest that a larger size DAF was present in tumor tissue of NSCLC and that the larger molecular weight was due to sialylation of the sugar moiety. In general, larger molecular weight glycoproteins are known as Warren-Glick phenomenon in cancer (32–35), a finding considered due to increased amounts of N-linked glycans by N-acetylglucosaminyltransferase (36). DAF has an N-linked sugar that is essential for its function (14), but it contributes little to the molecular weight. In the present study, neuraminidase treatment resulted in reduction of the molecular weight to 55 kDa. Based on the calculated mass of the peptide portion of DAF of 38 kDa, it is suggested that the molecular mass of DAF of 65 kDa in normal tissue consists of 42% total sugars, including 15% sialic acids, and that the molecular mass of DAF of 75 kDa in tumor lesions consists of 49% total sugars, including 27% sialic acids. This clearly indicates that sialylation in the O-linked sugar is responsible for the enlarged molecular weight of DAF in NSCLC tissue. It is unknown how the activity of DAF is modified by sialylation.

Using histochemical PNA staining, we previously reported that the expression of PNA-recognizing carbohydrates correlated with lymph node metastasis in human lung adenocarcinoma tissue (8). This observation implies that the net amount of PNA-binding carbohydrates increased in tumor lesions and correlated with the metastasis of tumor cells. At present, we have no evidence about whether the increased level of 55- to 65-kDa PNA-binding protein and/or DAF is responsible for the high frequency of metastasis in NSCLC because we observed a decrease of DAF in NSCLC and no correlation between PNA-binding protein/DAF level and clinicopathologic variables, including nodal involvement. In addition, in in vitro chemo-invasion assays, we observed that the metastatic activity of DAF-overexpressed PC14 cells was not statistically different from that of nontransfected cells (P = 0.058; n = 6). Thus, although DAF is one of the PNA-binding proteins on the surface of lung cells, its expression alone seems to have no significant effect on the metastatic property of NSCLC.

In conclusion, we report a new 55- to 65-kDa PNA-binding protein in normal lung tissue where the core protein was identified as DAF, CD55. In NSCLC tissue, DAF was down-regulated at the transcription level and processed to a larger molecule through sialylation of the sugar moiety. Our results indicate that the reduced expression and heavy sialylation of DAF was associated with basal pathology of NSCLC and that these alterations of DAF make this protein a potential marker for NSCLC.


   Acknowledgments
 
We thank Professor Tsukasa Seya (Hokkaido University, Sapporo, Japan) for valuable suggestions and discussions.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 5/06; revised 8/15/06; accepted 8/29/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
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