1 Developmental Biology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 131, Kagamiyama, Higashihiroshima, Hiroshima 739-8526 and 2 Hiroshima Tissue Regeneration Project, Hiroshima Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and Technology Corporation, Hiroshima Prefecture Institute of Industrial Science and Technology, 31032, Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan
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Abstract |
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Keywords: chimeric protein/collagenase/drug delivery/fusion protein/type III collagen
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Introduction |
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Collagen is one of the extracellular matrix molecules and shows a unique biochemical property to form insoluble fibers (Kadler et al., 1996). This fiber provides a scaffold for the attachment and the migration to cells (Yoshizato et al., 1981
; Utoh et al., 2000
). Collagen is also known to regulate the growth and differentiation of cells in the process of embryogenesis, regeneration and wound healing (Yoshizato et al., 1984
, 1985
, 1988a
; Nishikawa et al., 1987
; Adams and Watt, 1993
; Koide et al., 1993
; Koseki and Yoshizato, 1994
; Koseki et al., 1996
; Asahina et al., 1999a
, 1999b
). These properties have prompted researchers to use collagens as a biomaterial for clinically managing the wound healing (Yannas and Burke, 1980
; Koide et al., 1993
; Ramshaw et al., 1995
; Pachence, 1996
). The easy manipulability of collagen also accelerated its use in cosmetic surgery, tissue engineering and as a drug deliverer (Yannas and Burke, 1980
; Fujiwara et al., 1990
, 1991
; Koide et al., 1993
; Fujioka et al., 1995
; Ramshaw et al., 1995
; Pachence, 1996
; Maeda et al., 1999
; Ochiya et al., 1999
). Collagen is also widely used in cell culture as a natural substrate for cells because of its adhesive nature (Yoshizato et al., 1984
, 1985
, 1988b
; Nishikawa et al., 1987
). In addition, collagen is degraded gradually when implanted in the body (Burke et al., 1985
; Middelkoop et al., 1995
; Bailey, 2000
). This biodegradable nature of collagen has been a reason for collagens to be utilized as a drug-delivering biomaterial (Fujiwara et al., 1990
, 1991
; Fujioka et al., 1995
; Maeda et al., 1999
).
Recently, we have constructed a chimeric protein composed of EGF and type III collagen (EGF-3A1) (Hayashi et al., 2001). The chimeric protein can be immobilized on tissue culture dishes or incorporated into collagen fibrils without abolishing the EGF activity. This strongly suggests that this type of chimeric collagen is useful as a biocompatible, biodegradable and adhesive fibrous mitogen for a variety of purposes in the area of tissue engineering. Our previous study on the EGF-3A1 chimeric protein showed its adhesiveness of collagen, but did not show the biodegradability of the collagen. The collagen biodegradability is crucial for a potential drug deliverer wherein the drug is immobilized to the collagen. The present study was aimed at addressing whether the collagen-based cytokine chimeric protein shows the expected biodegradability upon collagenolysis and liberates the immobilized cytokine without abolishing the targeted cytokine's biological activity. We undertook this study utilizing the IL-2-type III collagen chimeric protein (IL2-3A1) as a clinically important potential drug deliverer. IL2-3A1 was prepared basically following the previous method for EGF-3A1 chimeric protein (Hayashi et al., 2001
). We could immobilize the IL2-3A1 on plastic dishes owing to the adhesive nature of the collagen region of the chimera as we could do with EGF-3A1. In addition, IL-2 was found to be liberated from the dishes upon collagenolysis by bacterial collagenase and the freed IL-2 was capable of stimulating the growth of T cells. Hence we strongly suggest that the IL2-3A1 chimeric protein might be utilized as an IL-2 deliverer whose T cell mitogenic activity can be liberated by a collagenolytic environment in living tissues.
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Materials and methods |
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Sf9 cells were obtained from the Riken Cell Bank (Tsukuba, Japan) and maintained at 28°C in Grace's insect medium (Gibco/BRL, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone, Logan, UT) (Tomita et al., 1995, 1997
, 1999
; Hayashi et al., 2001
). Cells of CTLL-2, an IL-2-dependent mouse cytotoxic T-lymphocyte cell line (Gillis and Smith, 1977
), were obtained from the Riken Cell Bank and were maintained at 37°C in RPMI 1640 (Gibco/BRL) supplemented with 10% (v/v) FBS and 100 units/ml of IL-2 (Chemicon, Temecula, CA). The CTLL-2 cells are known to require IL-2 for their growth (Gillis et al., 1978
). IL-2 was not added to the medium in the experiments wherein the mitogenic activity of recombinant proteins was assayed using CTLL-2 cells.
Construction of recombinant baculoviruses
We designed the chimeric protein (IL2-3A1) consisting of human IL-2 and human type III collagen (Figure 1). The recombinant baculovirus bearing the cDNA of IL2-3A1 was constructed as follows. The XhoI recognition site was introduced into IL-2 cDNA (a gift from Dr K.Koyama of Kitasato University School of Medicine, Kanagawa, Japan) at nucleotides 514519, the numbering being according to the reported sequence data (GenBank accession number NM_000586), immediately before the termination codon of IL-2 cDNA, using a Transformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA). The primer employed for this mutation was 5'-CTCAACACTAACTCGAGAATTAAGTGCTTCC-3', the bold letters indicating the mutated sequence. Two site-directed mutations were introduced in collagen pro
1(III) cDNA using the Transformer Site-Directed Mutagenesis Kit. One mutation was introduced in the sequence of the procollagen N-proteinase recognition site using mutagenesis primers of 5'-CAGAACTATTCTCTCCCGTATGATTCATATG-3', the bold letters indicating the mutation, to prevent the removal of IL-2 from IL2-3A1 by endogenous procollagen N-proteinase (Prockop and Kivirikko, 1995
). The other was the XhoI recognition site inserted at nucleotides 308313 of collagen pro
1(III) cDNA, the numbering being after Ala-Kokko et al. (Ala-Kokko et al., 1989
) (GenBank accession number X14420) using mutagenesis primers of 5'-GACATAATATGTGACGCTCGAGAATTAGACTGC-3', the bold letters representing the mutated sequence. cDNAs of type III procollagen and IL-2 were cleaved by XhoI and then ligated to each other by T4 DNA ligase. The resulting cDNA was inserted at the BamHI restriction site of the baculovirus transfer vector pAcYM1 (Matsuura et al., 1987
) (a gift from Dr Y.Matsuura of the National Institute of Infectious Diseases, Tokyo, Japan). The resultant vector was dubbed pAcIL2-3A1. A transfer vector for control experiments was also prepared by inserting IL-2 cDNA alone at the BamHI restriction site of pAcYM1. The resultant vector was dubbed pAcIL2. The recombinant transfer vectors (pAcIL2-3A1 and pAcIL2) and the linearized DNA of Autographa californica multinuclear polyhedrosis virus (AcMNPV) (Baculogold; Pharmingen, San Diego, CA) were co-transfected into Sf9 cells by lipofection to obtain recombinant baculoviruses, AcIL2-3A1 and AcIL2, respectively. The baculoviruses were isolated, purified and amplified as described previously (Tomita et al., 1995
, 1997
, 1999
).
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Sf9 cells in monolayer cultures were infected with AcIL2-3A1 and were incubated for 48 h in Grace's medium supplemented with 10% (v/v) FBS and 50 µg/ml ascorbic acid (Gibco/BRL) at 28°C (Tomita et al., 1995, 1997
, 1999
; Hayashi et al., 2001
). Proteins in the medium were precipitated with 33%-saturated ammonium sulfate for 16 h at 4°C. The precipitants were dissolved in 0.5 M acetic acid with or without 100 µg/ml pepsin (Sigma, St. Louis, MO) and incubated for 16 h at 4°C. The digests were lyophilized and dissolved in the sample buffer for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). All samples in SDSPAGE sample buffer were boiled for 5 min, cast on the gels of 0.1% SDS7.5% polyacrylamide and electrophoresed under reducing conditions. The gels were stained with Coomassie Brilliant Blue R-250 (CBB) or taken for Western blotting as follows. The proteins in the gels were electrotransferred to nitrocellulose filters (Schleicher and Schuell, Dassell, Germany) as described previously (Tomita et al., 1995
, 1997
, 1999
; Hayashi et al., 2001
). The membranes were reacted with anti-human type III collagen antibodies (LB-1300; LSL, Tokyo, Japan) or anti-human IL-2 antibodies (41202; Genzyme, Cambridge, MA) and visualized with a Vectastain ABC Kit according to the protocol of the manufacturer (Vector Laboratories, Burlingame, CA).
Assay of mitogenic activity of IL2-3A1
Sf9 cells were infected with AcIL2-3A1 or AcIL2 as above and cultured for 72 h. The culture media were collected, centrifuged and assayed for their mitogenic activity toward CTLL-2 cells as follows. A suspension of CTLL-2 cells in the IL-2-free medium was inoculated into wells of a 96-well plate at a density of 104 cells/well. The CTLL-2 cells were cultured for 24 h in the medium supplemented with the above Sf9 cell-culture media containing various concentrations of IL2-3A1 or IL-2. MTT reagent (Chemicon) was added to the media. After 4 h, the MTT formasan precipitates that formed in the wells were dissolved in 0.04 M HCl in 2-propanol. The absorbance of the contents of each well was measured at 570 nm.
Liberation of IL-2 region from IL2-3A1 by collagenase treatment
The IL2-3A1 chimeric proteins in Sf9 cell-culture media were precipitated with 33%-saturated ammonium sulfate as above, resuspended in 2 mM CaCl2 and 50 mM TrisHCl, pH 7.2, and treated for 2 h at 37°C with 100 units/ml of bacterial collagenase (chromatographically purified collagenase, form III, from Clostridium histolyticum; Advance Biofactures, Lynbrook, NY), the digestion being terminated by addition of an equal volume of double-strength SDSPAGE sample buffer. After being boiled for 5 min, the samples were subjected to electrophoresis on 520% acrylamide gradient gels (Pagel; Atto, Tokyo, Japan) followed by silver staining or Western blotting with the antibodies against human type III collagen or human IL-2 as described above.
Isolation and purification of IL2-3A1 from culture media of AcIL2-3A1-infected Sf9 cells
The IL2-3A1 chimeric proteins were precipitated from Sf9 cell-culture media with 33%-saturated ammonium sulfate as above, resuspended in 10 mM TrisHCl, pH 7.5, and 20 mM EDTA and were dialyzed against 10 mM TrisHCl, pH 7.5, and 20 mM EDTA at 4°C. The IL2-3A1 in this solution was purified using an ImmunoPure Protein G IgG Orientation Kit according to the protocol of the manufacturer (Pierce, Rockford, IL). A 9 mg amount of anti-human IL-2 antibodies (Genzyme) was bound to a protein G column and cross-linked by dimethyl pimelimidate. After being washed with 0.1 M glycineHCl, pH 2.8, to remove the unbound antibodies, the column was equilibrated with 10 mM TrisHCl, pH 7.5, and 20 mM EDTA. The proteins prepared as above were loaded on the column. The column was washed with 10 mM TrisHCl, pH 7.5, and the bound proteins were eluted with 0.1 M glycineHCl, pH 2.8, 1 ml of eluate being collected in tubes containing 50 µl of 1 M TrisHCl, pH 9.5. The eluates were analyzed by SDSPAGE followed by CBB staining as described above.
Immobilization of IL2-3A1 on the surface of tissue culture dishes
The solutions (200 µl) containing various concentrations of authentic IL-2 (Chemicon) or IL2-3A1 purified as above were placed in wells of 96-well plates and incubated for 16 h at 4°C to coat the wells with each of them. The wells were washed vigorously five times with phosphate-buffered saline (PBS) and blocked with 1% bovine serum albumin (BSA) for 4 h at 4°C. The immobilized proteins were quantified using a Quantikine Human IL-2 ELISA Kit (R & D Systems, Minneapolis, MN). The degrees of coating were estimated by measuring the absorbance of the contents of each well at 450 nm.
Liberation of IL-2 from immobilized IL2-3A1 by collagenase treatment
A solution containing 500 ng/ml of IL2-3A1 and 10% FBS was prepared by mixing 450 µl of the purified IL2-3A1 (3.3 µg/ml), 300 µl of FBS and 2250 µl of PBS. Aliquots (500 µl) of this solution were placed in each well of 24-well plates and incubated for 16 h at 4°C to coat the wells with IL2-3A1. Wells were also coated with 500 µl of the culture medium containing 500 ng/ml of IL2-3A1 wherein Sf9 cells (7.5x106 cells) transfected with AcIL2-3A1 were cultured in a volume of 15 ml for 3 days. The wells were washed vigorously five times with PBS. In the case of purified IL2-3A1, the presence of FBS was necessary in the coating procedure, because most of the IL2-3A1 proteins coated in its absence were spontaneously released from the dishes during the following cell growth assay (data not shown). Cell Culture Inserts with porous poly(ethylene terephthalate) membranes (pore size, 0.4 µm; Falcon 3495) were obtained from Becton Dickinson (Franklin Lakes, NJ) and were placed in the above IL2-3A1-coated wells. CTLL-2 cells suspended in the IL-2-free medium were inoculated into the Inserts at 2.5x104 cells/Insert. In this culture device, CTLL-2 cells could be cultured without directly contacting the coated IL2-3A1. CTLL-2 cells were allowed to grow for 2 days in the medium supplemented or not with 27 units/ml of bacterial collagenase. The growth of CTLL-2 cells was assayed using a Cell Counting Kit according to the protocol of the manufacturer (Dojindo, Kumamoto, Japan) (Ishiyama et al., 1993). The absorbance of the contents of each well was measured at 450 nm.
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Results |
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We constructed the IL2-3A1 chimeric protein consisting of human IL-2 and human type III collagen shown in Figure 1. The IL-2 was fused to the collagen at the outer side of the short triple helical domain in the N-propeptide of procollagen. In the normal biosynthesis, type III collagen is synthesized as larger precursor molecules known as procollagen. After the secretion of the procollagen from cells, the N- and C-propeptides of procollagen are cleaved by specific enzymes, called N- and C-proteinases, respectively (Prockop and Kivirikko, 1995
). Therefore, the IL-2 of IL2-3A1 might also be removed from IL2-3A1 by N-proteinase in cell culture medium. To prevent such extracellular IL-2 removal from the chimeric collagen, the amino acid sequence in the N-proteinase cleavage site was altered, in which Pro153Gln154 was changed to Leu153Pro154. Sf9 cells were infected with baculovirus vectors carrying IL2-3A1 cDNA and cultured for 3 days. The chimeric proteins in the culture medium were precipitated with 33%-saturated ammonium sulfate and analyzed by SDSPAGE under reducing conditions (Figure 2
). The gels were stained with CBB (Figure 2A
) or subjected to Western blotting with specific antibodies against human type III collagen (Figure 2B
) or human IL-2 (Figure 2C
). The cells infected with AcIL2-3A1 produced a band whose estimated molecular mass (Mr) was 150 K (Figure 2A
, IL2-3A1; this band is marked by `
' at the right side of the lane). This 150 K band was immuno-reactive with both anti-human type III collagen antibodies (Figure 2B
) and anti-human IL-2 antibodies (Figure 2C
). Hence it was concluded that the protein with an approximate mass of 150 K synthesized by AcIL2-3A1-infected Sf9 cells was the chimeric protein consisting of IL-2 and type III collagen. A faint immuno-reactive band was seen in the pepsin-untreated lane of Figure 2B
as marked by `
', its estimated molecular mass being 100 K. This band was thought to be a degradation product of IL2-3A1.
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We investigated whether IL2-3A1 fusion proteins retain the IL-2 activity. Sf9 cells were infected with AcIL2-3A1 or AcIL2 and cultured for 3 days. Their culture media were harvested and added to cultures of cytotoxic T lymphocyte cell line, CTLL-2, whose growth depends on IL-2 (Gillis et al., 1978). After 24 h, their growth was assayed using the MTT method. As a negative control experiment, the CTLL-2 cells were cultured in the medium supplemented with the culture supernatant of 3 day culture of non-infected Sf9 cells. No growth of CTLL-2 cells was observed in this medium (data not shown). As shown in Figure 3
, CTLL-2 cells grew depending on the concentration of IL2-3A1 added. The mitogenic activity of IL2-3A1 was almost equal to that of IL-2, indicating that IL-2 in the IL2-3A1 chimera retains its full biological activity. Hence it can be said that the fusion protein is not only structurally, but also functionally, chimeric, showing the normal helix formation of collagen and the mitogenic activity of IL-2.
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We tested whether the IL-2 region in IL2-3A1 is freed from the chimera by treating it with collagen-degrading enzymes. We utilized Clostridium histolyticum collagenase, which digests triple helical regions of collagen into small fragments (Mookhtiar and van Wart, 1992) as such an enzyme. IL2-3A1 proteins were treated with the collagenase and were separated on 520% acrylamide gradient gels under reducing conditions (Figure 4
). The gels were silver-stained (Figure 4A
) or subjected to Western blotting using anti-human type III collagen antibodies (Figure 4B
) or anti-human IL-2 antibodies (Figure 4C
). The IL2-3A1 not treated with collagenase was immunoreactive with both antibodies against human type III collagen (Figure 4B
, marked by `
') and human IL-2 (Figure 4C
, marked by `
'). The collagenase treatment removed the anti-IL-2 antibody-positive 150 K band (Figure 4C
) and, instead, a new anti-IL-2 antibody-positive band of lower molecular mass appeared (Figure 4C
, marked by `
'). This lower molecular mass band did not react with anti-human type III collagen antibodies. Hence we concluded that the IL-2 region in IL2-3A1 molecules can be liberated from the mother molecules by the collagenase treatment.
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We tested whether IL2-3A1 can be immobilized on plastic dishes. For this test, IL2-3A1 was purified from the ammonium sulfate precipitates of culture media of the chimeric collagen-producing Sf9 cells using an immuno-affinity column. As shown in Figure 5A, IL2-3A1 was highly purified to a single band on SDSPAGE gels. About 10 µg of purified IL2-3A1 were obtained from 400 ml of cultures. The purified IL2-3A1 was coated on tissue culture plates. As shown in Figure 5B
, the IL2-3A1 was immobilized on plastic in an IL2-3A1 concentration-dependent manner, whereas only a small amount of free IL-2 was adsorbed on the dishes.
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We investigated whether IL-2 can be freed from the immobilized IL2-3A1 without abolishing its biological activity when the collagen moiety of the immobilized chimera is degraded. To address this issue, we developed the following assay system for the measurement of the mitogenic activity of the liberated IL-2. The purified IL2-3A1 was coated on wells of 24-well plates. CTLL-2 cells were inoculated in Falcon Cell Culture Inserts. These Inserts were placed in the IL2-3A1-immobilized wells in such a way that the cells in the Inserts were immersed in the culture media in the wells. CTLL-2 cells did not make direct contact with the immobilized IL2-3A1 in this assay apparatus, but the cells would be exposed with IL-2 and would be mitotically stimulated if biologically active IL-2 is liberated from the immobilized IL2-3A1 by collagenolytic activity. Bacterial collagenase was supplemented to the culture medium in the wells and the growth of CTLL-2 cells was assayed by colorimetric assay (Figure 6).
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Discussion |
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The half-life of intravenously injected IL-2 is only 713 min in humans (Konrad et al., 1990). On the other hand, subcutaneously injected collagens remain non-degraded by endogenous interstitial collagenases for a few weeks (Burke et al., 1985
; Middelkoop et al., 1995
; Bailey, 2000
). Moreover, such a tightly coiled triple helical collagen molecule assembles into water-insoluble fibrils which are cleaved only by collagenases and are resistant to other proteinases even at the physiological temperature (Birkedal-Hansen et al., 1985
; Welgus et al., 1985
). These facts led us to postulate that molecules fused with collagen fibrils may prolong their half-life. Actually, it was reported that collagens are useful as a carrier of IL-2, BSA, interferon or DNA and prolong the half-life of these substances in the body (Fujiwara et al., 1990
, 1991
; Fujioka et al., 1995
; Maeda et al., 1999
; Ochiya et al., 1999
). Previously, we showed that EGF-collagen chimeric proteins can be incorporated into the collagen fibrils (Hayashi et al., 2001
), which suggests that IL2-3A1 might be able to be incorporated into the fibrils. This incorporation should prolong the half-life of the IL-2.
A local accumulation of IL-2 may cause inflammatory responses at the site of administration because IL-2 molecules play a central role in inflammatory response (Feghali and Wright, 1997). However, when IL-2 molecules are administered in the body using collagen as the carrier, IL-2 molecules show antitumor activity without inducing inflammatory responses (Fujiwara et al., 1990
, 1991
). Moreover, the degradation of collagen fibrils can be easily controlled by incorporating macromolecules such as gelatin and hyaluronic acid into the collagen matrices or by cross-linking of collagen fibrils by appropriate chemical reagents (Koide et al., 1993
; Middelkoop et al., 1995
; Bailey, 2000
). Therefore, IL-2 could be slowly released from the implanted fibrils in a controllable manner. The shielding effect of collagen and the controllability of the degradation rate of collagen may prevent IL-2 from inducing inflammation at the site of administration. Wrapping up the soluble IL-2 with collagen might be just as effective. However, we think that the chimerization of IL-2 and collagen has significant merit as compared with wrapping up, because it is likely that IL-2 in the chimeric protein can be maintained in the collagen gel more stably than soluble IL-2 wrapped with collagen because IL-2 is covalently bound to collagen. Thus, IL2-3A1 might act as an effective carrier of IL-2.
Generally, implanted collagen molecules in the body are degraded slowly by interstitial collagenase (Burke et al., 1985; Bailey, 2000
). Therefore, it is important to know whether the IL-2 region of the immobilized IL2-3A1 molecules can be liberated by collagenase without losing its biological activity. The major aim of the present study was to address this issue. We devised an in vitro model which mimics the liberation of IL-2 from the immobilized IL2-3A1 molecules upon in vivo collagenolysis. In this model, IL2-3A1 fusion proteins were coated on cell culture dishes in the presence of 10% FBS and CTLL-2 cells were cultured in Cell Culture Inserts to avoid the direct interaction between the immobilized IL2-3A1 and CTLL-2 cells. When bacterial collagenase was added to the medium, IL-2 was freed from the immobilized IL2-3A1. However, a significant amount of IL-2 activity was released from the immobilized surface without collagenase treatment (Figure 6I , B
), which is an unfavorable aspect of chimeric collagen as a drug deliverer. This unfavorable release of IL-2 activity could be significantly reduced when the IL2-3A1 in the culture medium of AcIL2-3A1 infected Sf9 cells was coated without further purification on cell culture dishes (Figure 6II, B
). However, a certain extent of the release was still observed. One of the reasons for this spontaneous release was thought to be that the immobilized chimeric collagen cannot form collagen fibrils on the coated surface. The chimeric collagen prepared in the present study contained the C-propeptide region, which has been known to inhibit the fibril formation of collagen molecules (Romanic et al., 1991
). Hence it is most likely that the IL2-3A1 chimeric collagen was coated on wells without forming fibrils. Although not tested in the present study, such a release could be substantially suppressed by mixing with a relatively excess amount of wild-type fibril-forming collagens or by incorporating the chimera into collagen gels. Such mixing of a small amount of IL2-3A1 has another merit. As mentioned above, the degradation rate of excess normal collagens or collagen gels can be controlled. In conclusion, it can be said that the IL-2 region of the immobilized IL2-3A1 is liberated, retaining its biological activity by collagenase digestion.
In the present study, we prepared the chimeric collagens with the C-propeptide region, because this region is necessary for the trimer formation of procollagen (McLaughlin and Bulleid, 1998). However, as described above, significant amounts of IL2-3A1 are spontaneously released from the wells. The removal of C-propeptide from IL2-3A1 might suppress this unfavorable release of IL-2 activity from the immobilized surface. Based upon the present results, we are undertaking to produce IL2-3A1 chimeras which lack the C-propeptide region by the method reported previously in the case of EGF-collagen chimeric proteins (Hayashi et al., 2001
) and, therefore, can form fibrils under physiological conditions.
The collagenase used in this experiment is of bacterial (Clostridium histolyticum) origin and digests triple helical regions of collagen into small fragments (Mookhtiar and van Wart, 1992). On the other hand, vertebrate interstitial collagenases cleave collagens at a single site located at about
of the distance from the carboxyl terminus and generate two
- and
-long triple helical fragments (Gross and Nagai, 1965
; Mookhtiar and van Wart, 1992
; Bailey, 2000
), suggesting that the IL-2 region liberated by bacterial collagenase may not be equal to that which is to be liberated by interstitial collagenase. Therefore, the activity of the IL-2 region liberated by interstitial collagenases might be different from that by bacterial collagenase. However, the IL-2 region in the
-long fragment produced by interstitial collagenases should be further degraded into small fragments in a similar manner as in IL2-3A1 attacked by bacterial collagenase, because it is known that the denaturation temperature of the
- and
-long fragments decreases and these fragments are readily attacked by non-specific proteases (Bailey, 2000
). In addition, the present study showed that IL-2 in the intact molecule of IL2-3A1 exhibits mitogenic activity as high as free IL-2 and the freed IL-2 exhibits mitogenic activity as high as IL2-3A1. These results indicate that the chimerization and the collagenolysis do not affect the IL-2 activity.
Recently, Somasundaram et al. showed that IL-2 can bind to interstitial collagens and collagen-bound IL-2 is less biologically active than equimolar doses of soluble IL-2 (Somasundaram et al., 2000). The IL-2 molecule plays a central role in inflammatory responses (Feghali and Wright, 1997
). The activity of matrix metalloproteinases (MMPs) is up-regulated during inflammation and extracellular matrices in the surrounding tissues are degraded most probably by these MMPs (Nagase and Woessner, 1999
; Bailey, 2000
; Schuppan and Hahn, 2000
). Therefore, it may be possible that, through the binding to collagen, IL-2 is sheltered in the collagen fibrils and is timely liberated by MMPs on the occasions when the activity of IL-2 is pathologically required, as, for example, in the case of inflammation. IL2-3A1 may mimic such an in vivo process of supplying IL-2 to the immuno-inflammatory cells. The activity of MMPs is also up-regulated in cancerous tissues (Okazaki et al., 1997
; Werb, 1997
; Nagase and Woessner, 1999
). Therefore, if fibrils of IL2-3A1 are implanted at the site of tumors, the fibrils might be degraded by collagenases secreted by these cancer cells and the IL-2 might be liberated as a biological active form. This liberated IL-2 could stimulate the antitumor immune responses of the cells surrounding the tumor tissues. Thus, IL2-3A1 might be a novel and potentially useful deliverer of IL-2, which maintains IL-2 without being attacked by the protease and diffusing under the normal conditions and liberates the IL-2 when and where IL-2 is pathologically needed.
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Notes |
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4 To whom correspondence should be addressed, at the first address. E-mail: kyoshiz{at}hiroshima-u.ac.jp
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ala-Kokko,L., Kontusaari,S., Baldwin,C.T., Kuivaniemi,H. and Prockop,D.J. (1989) Biochem. J., 260, 509516.[ISI][Medline]
Asahina,K., Utoh,R., Obara,M. and Yoshizato,K (1999a) Matrix Biol., 18, 89103.[CrossRef][ISI][Medline]
Asahina,K., Obara,M. and Yoshizato,K. (1999b) Dev. Dyn., 216, 5971.[CrossRef][ISI][Medline]
Bailey,A.J. (2000) Wound Repair Reg., 8, 512.[CrossRef][ISI][Medline]
Birkedal-Hansen,H., Taylor,R.E., Bhown,A.S. Katz,J., Lin,H.-Y. and Wells,B.R. (1985) J. Biol. Chem., 260, 1641116417.
Burke,K.E., Naughton,G. and Cassai,N. (1985) Ann. Plast. Surg., 14, 515522.[ISI][Medline]
Devos,R., Plaetinck,G., Cheroutre,H., Simons,G., Degrave,W., Tavernier,J., Remaut,E. and Fiers,W. (1983) Nucleic Acids Res., 11, 43074323.[Abstract]
Feghali,C.A. and Wright,T.M. (1997) Front. Biosci., 2, 1226.
Fujioka,K., Takada,Y., Sato,S. and Miyata,T. (1995) J. Controlled Release, 33, 307315.[CrossRef][ISI]
Fujiwara,T., Sakagami,K., Matsuoka,J., Shiozaki,S., Uchida,S., Fujioka,K., Takada,Y., Onoda,T. and Orita,K. (1990) Cancer Res., 50, 70037007.[Abstract]
Fujiwara,T., Sakagami,K., Matsuoka,J., Shiozaki,S., Fujioka,K., Takada,Y., Uchida,S., Onoda,T. and Orita,K. (1991) Biotherapy, 3, 203209.[ISI][Medline]
Gillis,S. and Smith,K.A. (1977) Nature, 268, 154156.[ISI][Medline]
Gillis,S., Ferm,M.M., Ou,W. and Smith,K.A. (1978) J. Immunol., 120, 20272032.[Abstract]
Gross,J. and Nagai,Y. (1965) Proc. Natl Acad. Sci. USA, 54, 11971204.[ISI][Medline]
Hayashi,M., Tomita,M. and Yoshizato,K. (2001) Biochim. Biophys. Acta, 1528, 187195.[ISI][Medline]
Ishiyama,M., Shiga,M., Sasamoto,K., Mizoguchi,M. and He,P. (1993) Chem. Pharm. Bull., 41, 11181122.[ISI]
Kadler,K.E., Holmes,D.F., Trotter,J.A. and Chapman,J.A. (1996) Biochem. J., 316, 111.[ISI][Medline]
Kaplan,D.R. (1994) J. Chromatogr. B, 662, 315323.[CrossRef][Medline]
Koide,M., Osaki,K., Konishi,J., Oyamada,K., Katakura,T., Takahashi,A. and Yoshizato,K. (1993) J. Biomed. Mater. Res., 27, 7987.[ISI][Medline]
Konrad,M.W., Hemstreet,G., Hersh,E.M., Mansell,P.W.A., Mertelsmann,R., Kolitz,J.E. and Bradley,E.C. (1990) Cancer Res., 50, 20092017.[Abstract]
Koseki,N. and Yoshizato,K. (1994) Cell Adhes. Commun., 1, 355366.[ISI][Medline]
Koseki,N., Sato,H. and Yoshizato,K. (1996) Cell Adhes. Commun., 3, 463474.[ISI][Medline]
Lamberg,A., Helaakoski,T., Myllyharju,J., Peltonen,S., Notbohm,H., Pihlajaniemi,T. and Kivirikko,K.I. (1996) J. Biol. Chem., 271, 1198811995.
Luross,J.E. and Williams,N.A. (2001) Immunology, 103, 407416.[CrossRef][ISI][Medline]
Maas,R.A., Dullens,H.F.J. and Otter,W.D. (1993) Cancer Immunol. Immunother., 36, 141148.[ISI][Medline]
Maeda,M., Tani,S., Sano,A. and Fujioka,K. (1999) J. Controlled Release, 62, 313324.[CrossRef][ISI][Medline]
Matsuura,Y., Possee,R.D., Overton,H.A. and Bishop,D.H.L. (1987) J. Gen. Virol., 68, 12331250.[Abstract]
McLaughlin,S.H. and Bulleid,N.J. (1998) Matrix Biol., 16, 369377.[CrossRef][ISI][Medline]
Middelkoop,E., de Vries,H.J.C., Ruuls,L., Everts,V., Wildevuur,C.H.R. and Westerhof,W. (1995) Cell Tissue Res., 280, 447453.[CrossRef][ISI][Medline]
Mookhtiar,K.A. and van Wart,H.E. (1992) Matrix, Suppl., 1, 116126.[Medline]
Morgan,D.A., Ruscetti,F.W. and Gallo,R. (1976) Science, 193, 10071008.[ISI][Medline]
Myllyharju,J., Lamberg,A., Notbohm,H., Fietzek,P.P., Pihlajaniemi,T. and Kivirikko,K.I. (1997) J. Biol. Chem., 272, 2182421830.
Nagase,H. and Woessner,J.F.,Jr. (1999) J. Biol. Chem., 274, 2149121494.
Nishikawa,A., Taira,T. and Yoshizato,K. (1987) Exp. Cell Res., 171, 164177.[ISI][Medline]
Notbohm,H., Nokelainen,M., Myllyharju,J., Fietzek,P.P., Müller,P.K. and Kivirikko,K.I. (1999) J. Biol. Chem., 274, 89888992.
Ochiya,T., Takahama,Y., Nagahara,S., Sumita,Y., Hisada,A., Itoh,H., Nagai,Y. and Terada,M. (1999) Nature Med., 5, 707710.[CrossRef][ISI][Medline]
Okazaki,I. et al. (1997) Hepatology, 25, 580584.[ISI][Medline]
Pachence,J.M. (1996) J. Biomed. Mater. Res., 33, 3540.[CrossRef][ISI][Medline]
Prockop,D.J. and Kivirikko,K.I. (1995) Annu. Rev. Biochem., 64, 403434.[CrossRef][ISI][Medline]
Ramshaw,J.A.M., Werkmeister,J.A. and Glattauer,V. (1995) Biotechnol. Genet. Eng. Rev., 13, 335382.[ISI]
Romanic,A.M., Adachi,E., Kadler,K.E. Hojima,Y. and Prockop,D.J. (1991) J. Biol. Chem., 266, 1270312709.
Rosenberg,S.A. et al. (1985) N. Engl. J. Med., 313, 14851492.[Abstract]
Rosenberg,S.A. et al. (1987) N. Engl. J. Med., 316, 889897.[Abstract]
Schuppan,D. and Hahn,E.G. (2000) Gut, 47, 1214.
Siegel,J.P. and Puri,R.K. (1991) J. Clin. Oncol., 9, 694704.[Abstract]
Smith,K.A. (1988) Science, 240, 11691176.[ISI][Medline]
Snellman,A., Keränen,M.-R., Hägg,P.O., Lamberg,A., Hiltunen,J.K., Kivirikko,K.I. and Pihlajaniemi,T. (2000) J. Biol. Chem., 275, 89368944.
Somasundaram,R., Ruehl,M., Tiling,N., Ackermann,R., Schmid,M., Riecken,E.O. and Schuppan,D. (2000) J. Biol. Chem., 275, 3817038175.
Taniguchi,T., Matsui,H., Fujita,T., Takaoka,C., Kashima,N., Yoshimoto,R. and Hamuro,J. (1983) Nature, 302, 305310.[ISI][Medline]
Tomita,M., Ohkura,N., Ito,M., Kato,T., Royce,P.M. and Kitajima,T. (1995) Biochem. J., 312, 847853.[ISI][Medline]
Tomita,M., Kitajima,T. and Yoshizato,K. (1997) J. Biochem., 121, 10611069.[Abstract]
Tomita,M., Yoshizato,K., Nagata,K. and Kitajima,T. (1999) J. Biochem., 126, 11181126.[Abstract]
Trentham,D.E., Townes,A.S. and Kang,A.H. (1977) J. Exp. Med., 146, 857868.[Abstract]
Utoh,R., Asahina,K., Suzuki,K., Kotani,K., Obara,M. and Yoshizato,K. (2000) Dev. Growth Differ., 42, 571580.[CrossRef][ISI][Medline]
van der Rest,M. and Garrone,R. (1991) FASEB J., 5, 28142823.
Welgus,H.G., Burgeson,R.E., Wootton,J.A.M., Minor,R.R., Fliszar,C. and Jeffrey,J.J. (1985) J. Biol. Chem., 260, 10521059.
Werb,Z. (1997) Cell, 91, 439442.[ISI][Medline]
Yannas,I.V. and Burke,J.F. (1980) J. Biomed. Mater. Res., 14, 6581.[ISI][Medline]
Yoshizato,K., Obinata,T., Huang,H., Matsuda,R., Shioya,N. and Miyata.T. (1981) Dev. Growth Differ., 23, 175184.[ISI]
Yoshizato,K., Taira,T. and Shioya,N. (1984) Ann. Plast. Surg., 13, 914.[ISI][Medline]
Yoshizato,K., Taira,T. and Yamamoto,N. (1985) Biomed. Res., 6, 6171.[ISI]
Yoshizato,K., Makino,A. and Nagayoshi,K. (1988a) Biomed. Res., 9, 3345.[ISI]
Yoshizato,K., Nishikawa,A. and Taira,T. (1988b) J. Cell Sci., 91, 491499.[Abstract]
Received August 29, 2001; revised November 28, 2001; accepted December 26, 2001.