©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of a Novel Collagen-binding Protein from the Mushroom, Hypsizigus marmoreus, Which Inhibits the Lewis Lung Carcinoma Cell Adhesion to Type IV Collagen (*)

(Received for publication, September 29, 1994; and in revised form, November 14, 1994)

Kazutaka Tsuchida Yutaka Aoyagi (1) Shoji Odani (2) Takashi Mita Mamoru Isemura (§)

From the  (1)Department of Food and Nutritional Sciences, University of Shizuoka, Yada, Shizuoka 422, the Department of Third Internal Medicine, Niigata University School of Medicine, Niigata 951, and the (2)Department of Biology, Niigata University College of Science, Niigata 950, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A type IV collagen-binding protein of 23 kDa was isolated from the mushroom, Hypsizigus marmoreus. This protein, HM 23, bound to type IV and type I collagens and gelatin, and to much lesser extent to fibronectin, but not to laminin or bovine serum albumin. The adhesion of Lewis lung carcinoma cells was inhibited when the type IV collagen substratum was pretreated with HM 23. A computer search of the determined partial amino acid sequence indicated no homologous proteins reported. These results indicate that HM 23 is a hitherto undescribed fungus protein that can interact with animal extracellular matrix proteins.


INTRODUCTION

Adhesive interaction between metastatic tumor cells and the endothelial basement membrane is believed to be an important step in the blood-borne metastatic process(1, 2) . The basement membranes are composed of several adhesive proteins including laminin, type IV collagen, and occasionally fibronectin(3, 4) . A laminin peptide, YIGSR, has been reported to inhibit the lung tumor colony formation in mice injected with melanoma cells, presumably by blocking the adhesion of the cells to the basement membranes, since the peptide inhibits cell adhesion and migration toward laminin(5) . A protein kinase C inhibitor, MDL 27,032, inhibited adhesion of melanoma cells to type IV collagen and fibronectin and could reduce experimental metastasis of these cells(6) . The fibronectin-related peptides inhibiting the cell adhesion to fibronectin could interfere with tumor metastasis(7, 8, 9) . Moreover, metastasis was inhibited by the antibodies, which could inhibit the adhesion of tumor cells to an artificial reconstituted basement membrane, Matrigel(10) . These data suggest that inhibition of the tumor cell adhesion to basement membrane components has a potential use in preventing metastasis.

We have reported that catechins with specific chemical structures could inhibit the Lewis lung carcinoma cell adhesion to the endothelial cells (11) . More recent experiments have shown that these catechins inhibit the adhesion of the tumor cells to fibronectin and laminin. (^1)One of these catechins, (-)-epigallocathechin gallate, has been shown to inhibit experimental metastasis of Lewis lung carcinoma cells(12) . Catechins are green tea constituents, and these findings have led us to examine various food extracts to test if they inhibit tumor cell adhesion to the basement membrane components. We have found that extracts of certain mushrooms have such an activity. In the present paper, we describe the isolation of a protein that binds to type IV and type I collagens and its inhibitory activity for the tumor cell adhesion to type IV collagen.


EXPERIMENTAL PROCEDURES

Materials

The edible mushroom, Hypsizigus marmoreus(13) , was obtained from a local market. Type IV collagen (from bovine crystalline lens, Nitta-gelatin Co., Osaka, Japan) was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc., Tokyo) at a concentration of 1 mg/ml wet gel. Fibronectin (from human plasma) and laminin (from mouse Engelbreth-Holm-Swarm sarcoma) were purchased from Iwaki-glass Co., Tokyo, Japan. MTT (^2)was from Chemicon International Inc., and serum-free culture medium Cosmedium 001 was from Cosmo Bio Co. Ltd. (Tokyo).

Isolation of a Type IV Collagen-binding Protein from H. marmoreus

All purification steps were performed at 0-4 °C. The mushroom, H. marmoreus (100 g) was homogenized in 200 ml of PBS containing 10 mM 6-aminohexanoic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine hydrochloride, and 1.3 mM EDTA (buffer A). The homogenate was centrifuged at 9,000 rpm for 10 min. To the supernatant was added ammonium sulfate to give 70% saturation, and the precipitated proteins were separated by centrifugation at 20,000 rpm for 10 min. The precipitates were dissolved in buffer A and dialyzed against the same buffer.The non-diffusible H. marmoreus protein fraction was applied onto a column (3.5 times 4 cm) of Sepharose 4B pre-equilibrated with buffer A. The unbound fraction was then loaded onto a column (1.5 times 3 cm) of type IV collagen-Sepharose 4B equilibrated with buffer A. The column was washed with buffer A and subsequently with 1 M NaCl in buffer A. The bound fraction was then eluted with 4 M urea, 1 M NaCl in buffer A, and fractions were collected. Each fraction was monitored by SDS-PAGE as described previously(14) , and fractions containing 23-kDa protein were collected and dialyzed against PBS. The type IV collagen-binding protein thus obtained was designated as HM 23.

Protein was determined by protein assay (Bio-Rad Laboratories K.K., Tokyo) using BSA as standard, and amino acid composition was determined using a Hitachi 835 amino acid analyzer as described previously(15) . Gel filtration was performed in PBS to determine the molecular size on a column (1.7 times 29.5 cm) of Superose 6 (Pharmacia Biotech Inc.) equipped with an LKB Ultrochrom GTi HPLC separation system (LKB Japan Co. Ltd., Tokyo). The elution positions of HM 23 and reference proteins (thyroglobulin, apoferritin, BSA, alpha-chymotrypsin, and lysozyme) were monitored by absorbance at 280 nm.

Amino Acid Sequences of Lysyl Endopeptidase Peptides

Lyophilized HM 23 was dissolved in 6 M guanidine and 5 mM EDTA in 1 M Tris buffer (pH 7.5). The solution was incubated at 55 °C for 1 h and then subjected to proteolysis with lysyl endopeptidase (enzyme/substrate ratio, about 1/100 by weight) at 37 °C for 3 h. The peptides generated were separated by HPLC on a column of TSK-GEL (type ODS-120T, 0.45 times 25 cm), and their amino acid sequences were determined by using a sequencer as described previously(16) .

Immunological Methods

Polyclonal antibody against HM 23 was prepared by alternative intraperitoneal injections of the antigen with Freund's complete adjuvant into BALB/c mice. Immunoblotting was performed as described previously(15) . To examine the interaction between HM 23 and various proteins, solid phase enzyme-linked immunoassays were performed using anti-HM 23 polyclonal antibody as described previously(15) . Briefly, plastic 96-well multidishes were coated with 20 µg/ml type IV and I collagens, gelatin, fibronectin, laminin, or BSA. The plates were then incubated with serial 2-fold dilutions of HM 23 in buffer A containing 0.05% Tween 20 starting at a protein concentration of 50 µg/ml. The bound HM 23 was determined with anti-HM 23 polyclonal antibody as the first antibody and with peroxidase-conjugated anti-mouse IgG as the second antibody, and absorbance was measured at 492 nm after the reaction with the o-phenylenediamine/H(2)O(2) substrate.

To examine the possibility that HM 23 was bound to type IV collagen as a lectin, competitive inhibition by possible hapten saccharides was examined using anti-HM 23 polyclonal antibody. The plastic multidishes precoated with type IV collagen were incubated with 50 µg/ml HM 23 in the presence of methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, lactose, or N-acetyl-D-glucosamine at 250 mM in buffer A. The bound HM 23 was then determined with anti-HM 23 as the first antibody and peroxidase-conjugated anti-mouse IgG as the second antibody as described above.

Effect of HM 23 on Adhesion of 3LL Cells to Type IV Collagen

Mouse Lewis lung carcinoma 3LL cells were cultured and maintained as described previously(11, 17) . A plastic 48-well multidish was coated with type IV collagen (20 µg/ml) in DMEM at 37 °C for 30 min in a humidified CO(2) incubator. After washing 3 times with DMEM, the wells were blocked with 1% BSA in DMEM at 37 °C for 1 h and then washed 3 times with DMEM. An HM 23 solution (100 µl) at various concentrations (35, 20, 10, 5, and 2.5 µg/ml in Cosmedium 001) was added to each well and incubated at 37 °C for 1 h. After washing 3 times with Cosmedium 001, a suspension of freshly trypsinized 3LL cells (4 times 10^4 in 100 µl of Cosmedium 001) was added to each well. After incubation at 37 °C for 2 h, non-attached cells were removed by washing with Cosmedium 001, and attached cells were examined microscopically.

For quantitative analysis of 3LL cell adhesion, an aliquot of an MTT reagent (10 µl) was added to each washed well, and absorbance was measured at 570 nm after incubation at 37 °C for 3 h as described previously(11, 17) .

Gelatinase Activity

This protease activity was examined by zymography with gels containing gelatin as described previously(18) .


RESULTS AND DISCUSSION

The presence of type IV collagen-binding protein(s) in the mushroom, H. marmoreus, was suggested by the observation that the number of 3LL cells attached to the type IV collagen-coated wells was greatly reduced when the wells had been treated with a protein fraction of its extract. When the protein fraction was subjected to affinity chromatography with type IV collagen-Sepharose 4B, SDS-PAGE indicated the presence of a single protein band of 23 kDa in the fractions eluted with 4 M urea, 1 M NaCl (Fig. 1). When BSA-Sepharose 4B was used as affinity gel, no such protein was detected (data not shown). The type IV collagen-binding protein, HM 23, thus obtained was dialyzed against PBS, and analysis of the concentrated solution revealed its homogeneity (Fig. 1). Isolated HM 23 was again bound by type IV collagen-Sepharose 4B quantitatively under the conditions used (data not shown), indicating that HM 23 bound directly to type IV collagen.


Figure 1: A, SDS-PAGE of fractions of affinity chromatography with type IV collagen-agarose. Lane1, marker proteins (BSA, ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, trypsinogen, soybean trypsin inhibitor, alpha-lactalbumin); lane2, H. marmoreus protein fraction; lane3, pass-through fraction; lanes4-12, fraction numbers of 1, 3, 5, 7, 10, 15, 30, 50, and 60, respectively, eluted with 4 M urea, 1 M NaCl. B, Western blot analysis. Lanes1 and 6, marker proteins; lanes2 and 4, H. marmoreus protein fraction; lanes3 and 5, HM 23. Lanes 1-3 were stained with Coomassie Brilliant Blue R-250, and lanes 4-6 were immunostained with anti-HM 23 antiserum.



Comparison of the electrophoretic properties of HM 23 under reducing and nonreducing conditions suggested that this protein was a single polypeptide without subunits linked by a disulfide bond (data not shown). The result of gel filtration through Superose 6 also indicated that HM 23 was a single polypeptide of 23 kDa (data not shown).

The amino acid composition is listed in Table 1. The amino acid sequence analysis of HM 23 resulted in no detection of phenylthiohydantoins, suggesting that the amino terminus of the protein is blocked. Therefore, the protein was subjected to lysyl endopeptidase digestion. The digest was then separated by HPLC (Fig. 2), and the sequences of three peptides were successfully determined (Table 2). A computer homology search has indicated that no homologous sequences have been reported yet, suggesting that HM 23 is a novel protein. However, it should be noted that the determined sequence of HM 23 has a structure homologous to that of human fibromodulin(19) , a collagen-binding protein that regulates the formation of collagen fibrils, when the frameshift was taken into account (Table 2).




Figure 2: HPLC of lysyl endopeptidase digests of HM 23 on TSK-GEL in 0.1% trifluoroacetic acid with a linear gradient of 1-48% acetonitrile in 60 min. Flow rate, 1 ml/min.





The immunoblotting analysis showed that the polyclonal antibody obtained was monospecific to HM 23 (Fig. 1). The results of the solid phase enzyme-linked immunoassay using the obtained antibody indicated that HM 23 was effectively and concentration dependently bound to the solid phase type IV collagen, type I collagen, and gelatin, and very weakly to fibronectin, but not to laminin or BSA (Fig. 3).


Figure 3: Enzyme-linked immunoassay for HM 23 binding to extracellular matrix proteins. Wells of multidishes were coated with 20 µg/ml type IV collagen (bullet), type I collagen (circle), gelatin (), fibronectin (box), laminin (), or BSA (). After incubation with serial 2-fold dilutions of HM 23 starting at 50 µg/ml, bound HM 23 was determined by using anti-HM 23 antiserum.



Since type IV collagen has carbohydrate side chains (20) including the glucosylgalactosyl unit linked to the hydroxylysine residue(21) , the possibility was examined that HM 23 was bound to type IV collagen as a lectin. The result revealed that inhibition of the binding with hapten saccharides, in any case, was less than 10%, indicating that a carbohydrate moiety is not involved in the binding. However, the involvement of other carbohydrate structures not testable by the hapten saccharides employed here could not be ruled out, and further work will be necessary to ask especially the question whether or not hydroxylysine-linked saccharides (21) are involved in this binding.

To examine the inhibition of the 3LL cell adhesion to type IV collagen by HM 23, plastic dishes precoated with type IV collagen at 20 µg/ml were incubated with HM 23 solutions at various concentrations. Newly trypsinized 3LL cells were added, and then the number of the adhered 3LL cells was examined microscopically. HM 23 at 35 µg/ml exhibited the distinct inhibitory activity for 3LL cell adhesion (Fig. 4). The efficiency of this inhibition was determined after the reaction with MTT to quantitate the attached cells. The percentage inhibitions were 22.8, 40.3, 65.3, 72.0, and 83.2% as mean values of three determinations with standard deviations of less than 10% at HM 23 concentrations of 2.5, 5, 10, 20, and 35 µg/ml, respectively. Thus, the adhesion of 3LL cells to type IV collagen was inhibited by HM 23 concentration dependently, and 50% inhibition was achieved at a concentration of about 7 µg/ml. On the other hand, adhesion of 3LL cells to fibronectin was not inhibited by HM 23 at concentrations up to 100 µg/ml. 3LL cells did not adhere to the wells coated with HM 23 at 35 µg/ml.


Figure 4: Phase contrast microscopy of 3LL cells cultured for 2 h on type IV collagen (A) or on type IV collagen pretreated with HM 23 at 35 µg/ml (B). Scale bar, 50 µm (B).



Collagenases are known to bind to collagen(22) . That the binding of HM 23 to collagens was not through enzyme-substrate interaction was suggested, since this activity was not detected in HM 23 by zymography in which at least six distinct gelatinase activities were detected with the Triton X-100 extract of 3LL cells (data not shown).

Although an inhibitor of the adhesion of metastatic cells to the basement membrane components such as type IV collagen may have potential use to prevent metastasis, direct application of HM 23 to in vivo experiments would be hampered by the host immune response. Thus, we need more studies in order to reveal whether or not the present results have relevance to the prevention of cancer metastasis. These include the determination of the structures involved in the binding between HM 23 and type IV collagen to find out an inhibitory peptide like GRGDSP and YIGSR(5, 7) .

There have been studies dealing with the interaction between animal extracellular matrix proteins and non-animal proteins(23, 24, 25, 26, 27, 28) . Some microorganisms such as Staphylococcus aureus and Candida albicans have fibronectin-binding proteins(23, 24) . There are also microorganisms that bind to collagen and laminin(23, 26, 27, 28) . These proteins may function in host tissue adherence. The relation between HM 23 and these proteins may be revealed once the primary structure of HM 23 is determined, although the amino acid sequences so far determined have suggested that there are no homologous proteins reported.

The biological significance of HM 23 is not known at present. It is known that certain kinds of mushrooms such as Cordyceps species (Tochukaso) grow on insects(29) . The association of a collagen-binding protein of mushrooms to collagen or collagen-related protein(s) of its host may have significance in host-hostess interaction.


FOOTNOTES

*
This investigation was supported in part by Special Coordination Funds of the Science and Technology Agency of the Japanese Government and a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of Shizuoka, Yada, Shizuoka 422, Japan. Tel.: 54-264-5531; Fax: 54-264-5099.

(^1)
K. Ogata, N. Mukae, Y. Suzuki, K. Satoh, N. Narumi, and M. Isemura, unpublished results.

(^2)
The abbreviations used are: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BSA, bovine serum albumin; DMEM, Dulbecco's minimal essential medium; PBS, 0.05 M phosphate-buffered saline containing 0.1 M NaCl (pH 7.5); PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.