U.S. patent application number 12/060056 was filed with the patent office on 2008-09-04 for method for cell adhesion and wound healing.
Invention is credited to In-San KIM, Jung-Eun KIM.
Application Number | 20080214463 12/060056 |
Document ID | / |
Family ID | 19668565 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080214463 |
Kind Code |
A1 |
KIM; In-San ; et
al. |
September 4, 2008 |
METHOD FOR CELL ADHESION AND WOUND HEALING
Abstract
The present invention relates to a method for cell adhesion and
wound healing with internal domains of .beta.ig-h3. Particularly,
the present invention relates to the method of using recombinant
proteins comprising one or more of 2.sup.nd or 4.sup.th internal
domain of .beta.ig-h3 for cell adhesion and wound healing, wherein
the 2.sup.nd or 4.sup.th internal domain of .beta.ig-h3 has
aspartic acid and isoleucine essential for interaction with
integrin which represent a high homology in base sequence of
.beta.ig-h3 internal domains. The recombinant proteins comprising
one or more 2.sup.nd or 4.sup.th internal domain of .beta.ig-h3 are
effective for cell adhesion and wound healing by itself and can be
used for developing cell culture medium and wound healing
agent.
Inventors: |
KIM; In-San; (Soosung-ku,
KR) ; KIM; Jung-Eun; (Soosung-ku, KR) |
Correspondence
Address: |
JHK LAW
P.O. BOX 1078
LA CANADA
CA
91012-1078
US
|
Family ID: |
19668565 |
Appl. No.: |
12/060056 |
Filed: |
March 31, 2008 |
Current U.S.
Class: |
514/9.4 ;
435/402; 530/324 |
Current CPC
Class: |
A61P 17/00 20180101;
A61K 38/00 20130101; A61P 17/02 20180101; A61P 43/00 20180101; C07K
14/78 20130101 |
Class at
Publication: |
514/12 ; 530/324;
435/402 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C07K 14/00 20060101 C07K014/00; C12N 5/06 20060101
C12N005/06; A61P 43/00 20060101 A61P043/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2000 |
KR |
2000-25662 |
Claims
1-15. (canceled)
16. A recombinant protein mediating cell adhesion comprising one or
more copies of the 4.sup.th fas-1 domain of .beta.ig-h3, wherein
the recombinant protein does not include full-length .beta.ig-h3
protein.
17. The recombinant protein according to claim 16, wherein the
4.sup.th fas-1 domain of .beta.ig-h3 consists of the amino acid
sequence 498-637 of human .beta.ig-h3 (SEQ ID NO: 26).
18. The recombinant protein according to claim 16, wherein the
recombinant protein comprises the amino acid sequence 502-637 of
human .beta.ig-h3 (SEQ ID NO:27) in quadraplicate.
19. The recombinant protein according to claim 16, wherein the
recombinant further comprises one or more copies of the 2 fas-1
domain of .beta.ig-h3.
20. A recombinant protein mediating cell adhesion comprising two or
more copies of the 2.sup.nd and/or the 4.sup.th fas-1 domain of
.beta.ig-h3.
21. The recombinant protein according to claim 20, wherein the
2.sup.nd fas-1 domain of .beta.ig-h3 consists of the amino acid
sequence 237-377 of .beta.ig-h3 (SEQ ID NO: 24).
22. The recombinant protein according to claim 20, wherein the
4.sup.th fas-1 domain of .beta.ig-h3 consists of the amino acid
sequence 498-637 of .beta.ig-h3 (SEQ ID NO: 26).
23. The recombinant protein according to claim 20, wherein the
recombinant protein comprises the amino acid sequence 502-637 of
.beta.ig-h3 (SEQ ID NO:27) in quadraplicate.
24. A recombinant protein consisting of one or more copies of the
2.sup.nd and/or 4.sup.th fas-1 domain of .beta.ig-h3.
25. The recombinant protein according to claim 24, wherein the
2.sup.nd fas-1 domain of .beta.ig-h3 consists of the amino acid
sequence 237-377 of .beta.ig-h3 (SEQ ID NO: 24).
26. The recombinant protein according to claim 24, wherein the
4.sup.th fas-1 domain of .beta.ig-h3 consists of the amino acid
sequence 498-637 of .beta.ig-h3 (SEQ ID NO: 26).
27. The recombinant protein according to claim 24, wherein the
recombinant protein consists of the amino acid sequence 502-637 of
.beta.ig-h3 (SEQ ID NO:27) in quadraplicate.
28. A wound healing agent comprising a therapeutically effective
amount of the recombinant protein of claim 16.
29. A wound healing agent comprising a therapeutically effective
amount of the recombinant protein of claim 20.
30. A wound healing agent comprising a therapeutically effective
amount of the recombinant protein of claim 24.
31. A solid support coated with the recombinant protein of claim
16.
32. A solid support coated with the recombinant protein of claim
20.
33. A solid support coated with the recombinant protein of claim
24.
Description
CONTINUING DATA
[0001] The present application is a U.S. national phase application
under 35 U.S.C. .sctn.371, of PCT/KR00/01428, filed Dec. 8,
2000.
FIELD OF THE INVENTION
[0002] The present invention relates to peptides for use in cell
adhesion and wound healing. More particularly, the present
invention relates to the use in cell adhesion and wound healing of
peptides containing one or more copies of the 2.sup.nd and/or
4.sup.th fas-1 domain of .beta.ig-h3, said 2.sup.nd and 4.sup.th
domains sharing a high homology in two amino acids, aspartic acid
and isoleucine, essential for binding to integrin and thus
mediating cell adhesion. Also, the present invention is concerned
with an expression system for the peptides useful in cell adhesion
and wound healing.
BACKGROUND OF THE INVENTION
[0003] .beta.ig-h3 is an extracellular matrix protein whose
expression is induced in various cell lines, including human
melanoma cells, mammary ephithelial cells, keratinocytes, and lung
fibroblasts, following signaling by active TGF-.beta. (Skonier, J.
et al., DNA Cell Biol. 13, 571, 1994). The ig-h3 gene was first
isolated by differential hybridization screening of a cDNA library
made from a human lung adenocarcinoma cell line that had been
treated with TGF-.beta.. .beta.ig-h3 gene encodes a 683-amino acid
protein that is highly conserved between species. It contains an
N-terminal secretory signal peptide and an Arg-Gly-Asp (RGD) motif
at the C-terminus. The RGD motif is found in many extracellular
matrix proteins modulating cell adhesion and serves as a ligand
recognition sequence for several integrins (Stonier, J. et al., DNA
Cell Biol., 11, 511, 1992).
[0004] According to several studies, .beta.ig-h3 is known to be
involved in cell growth and proliferation, wound healing, and cell
adhesion, although the underlying mechanisms for these functions
are still unclear. However, .beta.ig-h3 seems to play an important
role in the morphogenesis and interactions with cells and
extracellular matrix proteins in various tissues.
[0005] Some evidence related to the role of .beta.ig-h3 in
mediating cell attachment and detachment is provided by several
studies. For example, purified .beta.ig-h3 protein is found to
promote the attachment and spreading of skin fibroblasts while
inhibiting the adhesion of A549, HeLa and Wi-38 cells in serum-free
media. Particularly, .beta.ig-h3 is known to have inhibitory
activity against tumor cell growth, and to affect colony formation
and morphology. The inhibitory activity was demonstrated by the
report in which transfection of .beta.ig-h3 expression plasmids
into CHO (Chinese hamster ovary) cells led to marked decreases in
cell proliferation and the ability of these cells to form tumors in
nude mice. Further, a wound healing method was developed on the
basis of the finding that application of a pharmaceutically
effective amount of .beta.ig-h3 to wounds makes cells, especially
fibroblasts, spread over and adhere to the wound site.
Consequently, .beta.ig-h3, a cell adhesion molecule induced by
TGF-.beta. in various cell lines, plays a very important role in
cell growth, cell differentiation, wound healing, morphogenesis and
cell adhesion (Rawe, I. M. et al., Invest. Opthalmol. Vis. Sci. 38,
893, 1997; Lebaron, R. G. et al., J. Invest. Dermatol. 104, 844,
1995).
[0006] .beta.ig-h3 contains four 140 amino acid repeats with
internal homology, namely fas-1 domains. The internal repeat
domains have highly conserved sequences found in secretory proteins
or membrane proteins of various species, including mammals,
insects, sea urchins, plants, yeasts, and bacteria. Proteins
containing the conserved sequence are exemplified by periostin,
fasciclin I, sea urchin HLC-2, algal-CAM and mycobacterium MPB70.
The conserved domain in these proteins (hereinafter referred to as
"fas-1") consists of about 110 to 140 amino acids with two highly
conserved branches, H1 and H2, of about 10 amino acids each
(Kawamoto, T. et al., Biochem. Biophys. Acta. 1395, 288, 1998).
[0007] Four fas-1 domains are found in .beta.ig-h3, periostin, and
fasciclin I, two fas-1 domains in HLC-2, and only one fas-1 domain
in MPB70. Although the functions of the proteins are not elucidated
clearly, some of them are known to act as cell adhesion molecules.
For instance, .beta.ig-h3, periostin, and fasciclin 1 are reported
to mediate the adhesion of fibroblasts, osteoblasts, and nerve
cells, respectively. Also, it is disclosed that algal-CAM is a cell
adhesion molecule present in embryos of the algae Volvox (LeBaron,
R. G., et al., J. Invest. Dermatol. 104, 844, 1995; Horiuchi, K. et
al., J. Bone Miner. Res. 14, 1239, 1999; Huber, O. et al., EMBO J.
13, 4212, 1994).
[0008] At first, it was believed that the cell attachment activity
of .beta.ig-h3 would be mediated by the C-terminal RGD motif.
However, some research results revealed that the RGD motif is not
necessary for promoting the spreading of chondrocytes and that the
mature soluble .beta.ig-h3 whose RGD motif is deleted by
carboxyl-terminus processing is able to inhibit cell adhesion,
leading to the conclusion that the RGD motif of .beta.ig-h3 is
dispensable for mediating the cell attachment activity of
.beta.ig-h3. In addition, it has been recently reported that
.beta.ig-h3 promotes the spreading of fibroblasts via integrin
.alpha.1.beta.1 whereas the RGD motif of .beta.ig-h3 is not
necessary for mediating the cell adhesion property of .beta.ig-h3.
According to a recent report, .beta.ig-h3 binds specifically to
integrin to enhance the cell adhesion and spreading of cells
irrespective of RGD motif (Ohno, S. et al., Biochm. Biophys. Acta
1451, 196, 1999). Further, the conserved peptides H1 and H2 of
.beta.ig-h3 were found to have no influence on .beta.ig-h3-mediated
cell adhesion. These results, taken together, indicate that amino
acids indispensable for the cell attachment activity of .beta.ig-h3
exist somewhere other than the H1 and H2 regions. A computer search
based on homologies not only among the repeated fas-1 domains of
.beta.ig-h3 but also among fas-1 domains of other proteins revealed
that there are a few highly conserved amino acids in addition to H1
and H2 peptides, suggesting the possibility of the involvement of
the conserved amino acid sequences in the cell attachment
activity.
[0009] Of the domains of .beta.ig-h3, known to play an important
role in cell adhesion, either of the 2.sup.nd or 4.sup.th domain is
identified as a minimum domain essential for the cell adhesion of
the molecule in accordance with the present invention. Based on
these findings, recombinant proteins containing the essential
functional domains are also identified as being effective for wound
healing, in accordance with the present invention.
[0010] Recent research for wound healing has been subdivided into
cell biology and molecular biology and the promotion of wound
healing has had increasing applications in various clinical fields.
However, cell biological and molecular biological mechanisms of
wound healing still remain unclear. According to findings disclosed
thus far, wound healing is a tissue response to trauma, leading to
tissue repair through complex biological processes, including
chemotaxis, cell differentiation and replication, matrix protein
synthesis, angiogenesis, and wound reconstitution (Steed, D. L., et
al., Clin. Plast. Surg. 25, 397, 1998).
[0011] Growth factors are representative materials that appear in
the early stage of the wound healing process and control the
subsequent wound healing process. Having strong influence over all
stages of wound healing, growth factors act to control the growth,
differentiation and metabolism of cells and reorganize the environs
of the wound by their chemotactic properties which attract various
cells types that are involved in inflammation and tissue repair,
cellular proliferation, stimulating angiogenesis and the synthesis
and degradation of the extracellular matrix. PDGF (platelet-derived
growth factor) attracts fibroblasts to the wound and stimulates
them to proliferate, and transforming growth factor-beta
(TGF-.beta.) causes them to make collagen. PDGF is chemotactic for
most cells involved in wound healing, stimulates angiogenesis,
remodeling and contraction, and activates wound healing cells
(Mustoe, T. A. et al., J. Clin. Invest. 87, 694, 1991; Lepisto, J.
et al., J. Surg. Res. 53, 596, 1992). EGF (epidermal growth factor)
stimulates keratinocyte migration, angiogenesis and granulation
tissue development and activates mitogenesis of keratinocyets and
fibroblasts (Franklin, J. D. et al., Plast. Recsonst. Surg. 64,
766, 1979; Buckly, A. et al., Proc. Natl. Acad. Sci. USA, 82, 7340,
1985). bFGF (basic fibroblast growth factor) stimulates
angiogenesis, epithelialization, and collagenous fiber deposition,
and associates with heparin in various forms to perform relevant
functions (Tsuboi, R. et al., J. Exp. Med. 172, 245, 1990;
Kinsnorth, A. N. et al., Br. J. Surg. 77, 409, 1990). IGF
(insulin-like growth factor) enhances cell differentiation. VEGF
(vascular endothelial growth factor) increases vasopermeability and
promotes endothelial mitogenesis.
[0012] Of the growth factors and cytokines involved in wound
healing, TGF-.beta. is the most representative. Existing in three
forms (TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3) in mammals, the
cytokine plays important roles in the growth and differentiation of
various cells and has various complex functions, including control
of cell growth, regulation of immune responses, stimulation of
osteogenesis, induction of cartilage specific macromolecules, and
promotion of wound healing (Bennett, N. T. et al., Am. J. Surg.
165, 728, 1993). Appearing in the ephithelium during wound healing,
TGF-.beta. is believed to stimulate the expression of integrin
within keratinocytes during re-epithelialization. In recent
research into TGF-.beta. expression, it was revealed that
TGF-.beta.3 mRNA is expressed in the epithelia of normal skin and
acute and chronic wounds, while TGF-1 mRNA is not expressed in
normal skin and chronic wounds, but expressed in the epithelial
layer regenerated from acute wounds, and nowhere is expressed
TGF-.beta.2 mRNA (Schmid, P. et al., J. Pathol. 171, 191, 1993).
Based on the effects, even though their mechanisms are not firmly
established, TGF-.beta. is expected to play a major role in
re-epithelialization.
[0013] Expression of .beta.ig-h3 is up-regulated by TGF-.beta.,
suggesting that .beta.ig-h3 is involved in the mediation of some
signals of TGF-.beta.. CHO (Chinese hamster ovary) cells
transformed with .beta.ig-h3 expression plasmids are reported to
show decreased tumorigenic ability (Skonier, J. et al., DNA Cell
Biol. 13, 571, 1994). In contrast, .beta.ig-h3 expression is
down-regulated in dexamethasone-treated stem cells, some tumor
cells and the fibroblasts cultured from the skin lesion sites
afflicted with localized hyperostosis of melorheostosis.
.beta.ig-h3 is also reported to serve as a negative regulator of
osteogenesis (Genini, M. et al., Int. J. Cancer 66, 571, 1996;
Schenker, T. et al., Exp. Cell. Res. 239, 161, 1998; Kim, J. et
al., J. Cell Biochem. 77, 169, 2000). In addition to these
functions, .beta.ig-h3, known as a cell adhesion molecule, promotes
the adhesion and spreading of fibroblasts in the dermis. According
to studies into the distribution of .beta.ig-h3 in eye tissues, it
is reported that the adhesion molecule is expressed in corneal
epithelia of normal adults, intracorneal fetal stromal cells, and
the endothelial and stromal cells in the process of wound healing.
In addition, .beta.ig-h3 is expressed in the juxtaglomerular
apparatus and proximal tubules of the kidneys, and its expression
is increased in diabetes mellitus. Further, it is found in
subendothelial smooth muscles of the coronary arteries of normal
persons, and its amount is increased in the endometria of blood
vessels in the case of arteriosclerosis. However, the expression of
.beta.ig-h3 in normal dermal tissues and dermal wounds has not yet
been firmly established (Klintworth, G. K. et al., Am. J. Pathol.
152, 743, 1998; Munier, F. L. et al., Nature Genetics 15, 247,
1997; Streeten B. W. et al., Arch. Opthalmol. Vis. Sci. 38, 893,
1997). As mentioned above, the distribution and expression of
.beta.ig-h3 in normal human tissues remains unclear. Particularly,
there are no reports regarding expression patterns of .beta.ig-h3
in dermal wounds. However, some research groups have reported that
.beta.ig-h3 functions to promote the adhesion and spreading of
dermal fibroblasts, so that it is expected to make a contribution
to the promotion of wound healing.
SUMMARY OF THE INVENTION
[0014] With the background in mind, the intensive and thorough
research on .beta.ig-h3-mediated cell adhesion, leading to the
present invention, resulted in the finding that there exist highly
conserved amino acid sequences, in addition to H1 and H2 motifs,
among fas-1 domains of .beta.ig-h3 and among fas-1 domains of other
peptides, as analyzed by computer search, and particularly, high
homology is detected at aspartic acid and isoleucine residues at
positions near the H2 region. In addition, the 2.sup.nd and
4.sup.th domains of .beta.ig-h3, each containing the conserved
amino acid residues, were found to induce cell adhesion through
.alpha.3.beta.1 integrin. Further, recombinant proteins which were
designed to have the 2.sup.nd and/or 4.sup.th fas-1 domain of
.beta.ig-h3 were identified as being identical to wild type
.beta.ig-h3 in cell attachment and spreading activity and wound
healing effect.
[0015] Therefore, it is an object of the present invention to
provide peptides which contain conserved amino acid sequences
essential for cell attachment, spreading and detachment
activity.
[0016] It is another object of the present invention to provide the
use of the peptides in cell adhesion and wound healing.
[0017] It is a further object of the present invention to provide
an expression system for the peptides.
[0018] It is still a further object of the present invention to
provide a method for attaching cells.
[0019] It is still another object of the present invention to
provide a method for healing wounds.
[0020] In accordance with an aspect of the present invention, there
is provided a recombinant protein, comprising a portion of domains
of .beta.ig-h3, useful in mammalian cell attachment.
[0021] In accordance with another aspect of the present invention,
there are provided expression vectors p.beta.ig-h3 D-II,
p.beta.ig-h3 D-IV, and p.beta.ig-h3 D-IV 4X, capable of expressing
the 2.sup.nd and 4.sup.th fas-1 domain of .beta.ig-h3 corresponding
to amino acids 237-377 and 498-637, respectively.
[0022] In accordance with a further aspect of the present
invention, there are provided novel E. coli strains, transformed
with the expression vectors p.beta.ig-h3 D-II, p.beta.ig-h3 D-IV,
and p.beta.ig-h3 D-IV 4X, identified as E. coli BL21/His.beta.-g
(accession No. KCTC 0905BP), E. coli BL21/His.beta.-e (accession
No. KCTC 0904BP) and E. coli BL21/His.beta.-e4x (accession No. KCTC
0906BP), respectively.
[0023] In accordance with still a further aspect of the present
invention, there is provided a method for attaching cells,
comprising the steps of: preparing a recombinant protein containing
one or more copies of the 2.sup.nd and/or 4.sup.th domain of
.beta.ig-h3, by use of an expression vector; coating the
recombinant protein onto a solid support; and applying cells to the
protein-coated solid support.
[0024] In accordance with still another aspect of the present
invention, there is provided the use of the recombinant protein in
cell attachment.
[0025] In accordance with yet another aspect of the present
invention, there is provided the use of the recombinant protein in
wound healing.
[0026] In accordance with still yet another aspect of the present
invention, there is provided a method for healing wounds,
comprising the steps of: coating a solid support with a recombinant
protein containing one or more copies of the 2.sup.nd and/or the
4.sup.th domain of .beta.ig-h3; attaching skin cells to the solid
support; and applying the solid support to wounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1a is a schematic diagram showing recombinant proteins
.beta.igh3-WT and .beta.igh3-.DELTA.RGD, wherein conserved regions
are represented by and , and RGD motif by .RTM..
[0028] FIG. 2 is a photograph showing SDS-PAGE results of
recombinant proteins .beta.igh3-WT and .beta.igh3-.DELTA.RGD.
[0029] FIG. 3 is a microphotograph showing HCE cell adhesion and
spreading effects of recombinant proteins .beta.igh3-WT and
.beta.igh3-.DELTA.RGD after dying with crystal violet.
[0030] FIG. 4 shows curves in which the HCE cell adhesion and
spreading activities of the recombinant proteins .beta.igh3-WT and
.beta.igh3-.DELTA.RGD are found to be concentration-dependent as
measured by the count (A) and surface area (B) of attached
cells.
[0031] FIG. 5 shows histograms in which the HCE cell adhesion
activities of the recombinant proteins .beta.igh3-WT and
.beta.igh3-.DELTA.RGD are compared in terms of count (A) and
surface area (B) of attached cells.
[0032] FIG. 6a is a histogram showing effects of various compounds
on the HCE cell adhesion activities of the recombinant proteins
.beta.igh3-WT and .beta.igh3-.DELTA.RGD.
[0033] FIG. 6b is a histogram showing effects of divalent cations
on the HCE cell adhesion activities of the recombinant protein
.beta.igh3-WT.
[0034] FIG. 6c is a histogram showing the inhibition effect of
anti-integrin monoclonal antibody on the HCE cell adhesion activity
of the recombinant protein .beta.igh3-WT.
[0035] FIG. 6d is a histogram showing the inhibitory effect of
anti-integrin monoclonal antibody on the HCE cell adhesion
activities of various proteins.
[0036] FIG. 6e is a histogram showing adhesion specificity of K562
cells for the recombinant protein .beta.igh3-WT and matrix
proteins.
[0037] FIG. 7 is a schematic diagram showing recombinant proteins
having each of the fas-1 domains of .beta.ig-h3.
[0038] FIG. 8 is a photograph showing SDS-PAGE results of
recombinant proteins containing fas-1 domains of .beta.ig-h3.
[0039] FIG. 9 is a histogram showing HCE cell adhesion activities
of recombinant proteins containing fas-1 domains of
.beta.ig-h3.
[0040] FIG. 10 is a histogram showing the inhibitory effects of
anti-integrin antibodies on HCE cell adhesion activities of the
recombinant proteins containing fas-1 domains of .beta.ig-h3.
[0041] FIG. 11 shows parts of amino acid sequences of various
matrix proteins containing fas-1 domains.
[0042] FIG. 12 is a schematic diagram showing substitution mutants
of the 4.sup.th domain of .beta.ig-h3.
[0043] FIG. 13 is a photograph showing SDS-PAGE results of
recombinant substitution mutants of the 4.sup.th domain of
.beta.ig-h3.
[0044] FIG. 14 is a histogram showing cell adhesion activities of
substitution mutants of the 4.sup.th domain of .beta.ig-h3.
[0045] FIG. 15 is a schematic diagram showing recombinant proteins
.beta.igh3-D-1, .beta.igh3-D-IV 2X, 3X and 4X containing one, two,
three and four copies of the 4.sup.th domain of .beta.ig-h3.
[0046] FIG. 16 shows photographs of the recombinant proteins
.beta.igh3-D-IV, .beta.igh3-D-IV 2X, 3X and 4X run on 10% SDS-PAGE
(A) and 8% nondenaturing PAGE (B), which are purified with the aid
of Ni-NTA agarose resin
[0047] FIG. 17 shows optical photographs of wounds to which an
ointment base is applied alone (A) and in combination with
fibronectin (B), His-.beta.-b (C), and .beta.ig-h3-D-IV (D).
[0048] FIG. 18 shows microphotographs of wounds which are in the
process of re-epithelialization after being treated with an
ointment base alone (A) and in combination with fibronectin (B),
His-.beta.-b (C), and .beta.ig-h3-D-IV (D).
[0049] FIG. 19 shows optical photographs of wounds which have
collagenous fibers formed after being treated with an ointment
alone (A) and in combination with fibronectin (B), His-.beta.-b
(C), and .beta.ig-h3-D-IV (D).
[0050] FIG. 20 is a histogram showing HCE cell adhesion activities
of the recombinant proteins .beta.igh3-D-IV, .beta.igh3-D-IV 2X, 3X
and 4X, which contain at least one copy of the 4.sup.th domain of
.beta.ig-h3.
[0051] FIG. 21 shows optical photographs of wounds whose areas are
reduced after being treated with a chitosan base alone (A) and in
combination with fibronectin (B), .beta.ig-h3 3X (C), and
.beta.ig-h3 4X (D).
DETAILED DESCRIPTION OF THE INVENTION
[0052] In the present invention, recombinant proteins are prepared
on the basis of the 2.sup.nd and 4.sup.th fas-1 domains of
.beta.ig-h3 and used alone or in combination, for cell adhesion and
spreading. To select the 2.sup.nd and 4.sup.th domains, the domains
of .beta.ig-h3 active in cell adhesion and spreading was
identified. To this end, the C-terminal sequence Arg-Gly-Asp (RGD),
known as a ligand recognition sequence for several integrins, was
examined for its effect on the cell adhesion property of
.beta.ig-h3. The cell attachment activity was measured using the
number and surface area of attached cells. As a result, .beta.ig-h3
was found to promote cell adhesion and spreading, independent of
the RGD motif.
[0053] Based on this finding, chemical reagents were used to
address the specificity of cell adhesion activity of .beta.ig-h3
and to get further clues about the nature of the cell surface
receptor for .beta.ig-h3. The data obtained from the use of
chemical reagents suggest that the cell surface receptor for
.beta.ig-h3, which is involved in the cell adhesion activity of
.beta.ig-h3, could be one of the RGD-dependent integrins, which
require divalent cations for interaction with .beta.ig-h3.
[0054] Next, to identify minimum domains essential for the cell
adhesion function of .beta.ig-h3, an examination was made of the
ability of each fas-1 domain to mediate cell adhesion.
[0055] This examination was based on the fact that fas-1 domains
are found in various cell adhesion molecules, such as .beta.ig-h3,
periostin, fasciclin I, HLC-2, and algal-CAM and the number of
fas-1 domains present in such adhesion molecules varies from
protein to protein. This fact led to the inference that all four
fas-1 domains might not be required for the cell adhesion activity
of .beta.ig-h3 and in an extreme case, only one domain could
mediate the cell adhesion activity of .beta.ig-h3. In the present
invention, the either of 2.sup.nd or 4.sup.th fas-1 domain of
.beta.ig-h3 is revealed to be sufficient for the cell adhesion
function of .beta.ig-h3. These results lead to the conclusion that
the H1 and H2 sequences, common in the four domains of .beta.ig-h3,
are not essential for the mediation of the cell adhesion activity
of .beta.ig-h3. Additionally, two amino acids, that is, aspartic
acid and isoleucine at positions near the H2 region within the
2.sup.nd and 4.sup.th fas-1 domains, were found to be highly
conserved, implying that these amino acid residues constitute a
cell adhesion-related motif. The indispensability of the two
conserved amino acids for cell adhesion was identified using
substitution mutants of the 4.sup.th fas-1 domain of
.beta.ig-h3.
[0056] In another embodiment of the present invention, a wound
healing method is provided in which the 2.sup.nd and 4.sup.th fas-1
domains of .beta.ig-h3 are used, individually or in
combination.
[0057] An comparison was made of wound healing effects of mutant
.beta.ig-h3 proteins containing cell adhesion-active domains only
and those of wild type .beta.ig-h3 (.beta.ig-h3-WT) containing
domains a portion of the domains. In this regard, recombinant
proteins containing the cell adhesion-active domains were applied
to rats.
[0058] When a recombinant protein containing the 4.sup.th fas-1
domain of .beta.ig-h3 was used a pharmaceutically effective
ingredient for an ointment, wound shrinkage was observed, in
addition to re-epithelialization and collagenous fiber formation.
Ultimately, these results mean that one of the 2.sup.nd or 4.sup.th
fas-1 domain of .beta.ig-h3, in which the conserved aspartic acid
and isoleucine exist, is useful for wound healing and thus can be
utilized for the development of therapeutics for wounds.
[0059] Also, excellent cell adhesion and wound healing effects were
obtained using a recombinant protein containing the 2.sup.nd fas-1
domain of .beta.ig-h3 or 2.sup.nd and 4.sup.th domains.
[0060] Over the protein containing all of the domains, recombinant
proteins containing parts of the domains have an advantage in that
they can be produced in larger quantities because they are
synthesized in water-soluble forms and thus do not undergo
denaturation.
EXAMPLES
[0061] A better understanding of the present invention may be
obtained in light of the following examples which are set forth to
illustrate, but are not to be construed to limit the present
invention.
Example 1
Identification of Cell Adhesion Activity of RGD-Independent
.beta.ig-h3 Proteins
[0062] 1-1: Production of Recombinant .beta.ig-h3 Protein
[0063] In order to find the domains of .beta.ig-h3 which have, in
practice, cell adhesion and spreading activity, the C-terminal
terminal sequence Arg-Gly-Asp (RGD), known as a ligand recognition
sequence for several integrins, was examined for effect on the cell
adhesion property of .beta.ig-h3. In this regard, an RGD-deleted
recombinant .beta.ig-h3 protein (.beta.igh3-.DELTA.RGD) and a
wild-type recombinant .beta.ig-h3 protein (.beta.igh3-WT) were
prepared.
[0064] First, the full-length human .beta.ig-h3 cDNA cloned in
pBluescript (pBs.beta.ig-h3) was digested with NdeI and BglII. The
DNA fragment was subcloned into the EcoRV-EcoRI site of pET-29b(+)
(Novagen Inc.). .beta.igh3-WT was prepared by introducing a 1351 bp
NcoI fragment excised from .beta.ig-h3 cDNA into the NcoI site of
this clone. .beta.igh3-.DELTA.RGD was derived from .beta.igh3-WT by
cutting out a 3'-fragment of the .beta.igh3-WT plasmid with AoCI
and NotI followed by blunting and self ligation, as shown in FIG.
1.
[0065] After being transformed with each recombinant plasmid, E.
coli BL 21 DE3 was cultured in LB medium containing 50 .mu.g/ml
kanamycin at 37.degree. C. until the optical density (OD) at 595 nm
reached 0.5-0.6. The recombinant .beta.ig-h3 proteins were induced
using 1 mM isopropyl-.beta.-D-(-)-thiogalactopyranoside (IPTG) at
37.degree. C. for 3 hours. The pellet thus obtained was resuspended
in a lysis buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA,
1% Triton X-100, 1 mM PMSF, 0.5 mM DTT) and then sonicated. The
inclusion bodies were dissolved in a denaturation buffer of 8 M
urea containing 20 mM, followed by the purification of the
denatured proteins with the aid of Ni-NTA resin (Qiagen). The
recombinant proteins were eluted with 200 mM imidazole solution and
then dialyzed sequentially from high to low urea in 20 mM Tris-HCl
buffer containing 50 mM NaCl. These recombinant proteins were
analyzed using SDS-PAGE, as shown in FIG. 2.
1-2: Assay for the Cell Adhesion Activity of Recombinant
.beta.ig-h3 fas-1 Domain Proteins
[0066] Human corneal epithelial (HCE) cells used in this assay were
cultured in DMEM (EMEM/F-12, Gibco BRL) supplemented with 15% fetal
bovine serum, Gentamicin (40 .mu.g/ml), insulin (5 .mu.g/ml),
cholera toxin (0.1 .mu.g/ml) and human epidermal growth factor
(hEGF) at 37.degree. C. in 5% CO.sub.2.
[0067] The cell adhesion assay was performed as follows. First, the
recombinant .beta.ig-h3 proteins or other extracellular matrix
proteins were let to adhere to the bottoms of 96-well microculture
plates (Falcon) by incubation at 37.degree. C. for 1 hour and
blocked with PBS containing 0.2% BSA. The coated extracellular
matrix proteins were human plasma vitronectin (Promega), purified
human plasma fibronectin (pFN), chicken collagen types I and II
(Chemicon International Inc.), bovine collagen types IV and VI
(Chemicon), mouse laminin (Chemicon), and bovine serum albumin
(BSA) (Sigma). Cells were trypsinized and suspended in the culture
media at a density of 2.times.10.sup.5 cells/ml. 0.1 ml of the cell
suspension was added to each well of the plates coated with the
recombinant proteins.
[0068] Following incubation at 37.degree. C. for 1 hour, unattached
cells were removed by rinsing with PBS. Attached cells were
incubated for 1 hour at 37.degree. C. in 50 mM citrate buffer, pH
5.0, containing 3.75 mM p-nitrophenol-N-acetyl
1-.beta.-D-glycosaminide as a hexosaminidase substrate and 0.25%
Triton X-100, followed by the addition of 50 mM glycine buffer, pH
10.4, containing 5 mM EDTA to block the enzyme activity. A
measurement was made of absorbance at 405 nm in a Multiskan MCC/340
microplate reader.
[0069] To determine cell area as an index for cell adhesion
activity, 4.times.10.sup.4 cells were applied to substrates in
48-well culture plates. The attached cells were fixed with 8%
glutaraldehyde (Sigma) and then stained with 0.25% Crystal Violet
(Sigma) in 20% methanol. Measurement of cell areas was performed by
Image-Pro plus software (Media Cybernetics). Experiments were
repeated in triplicate with 200 or 300 measurements per site for
each experiment. Data is reported as the mean area at specific time
points .+-.standard error of mean.
[0070] As a result of the measurement of cell adhesion and
spreading activity using .beta.igh3-WT and .beta.igh3-.DELTA.RGD,
the numbers and surface areas of HCE cells which adhered to
.beta.igh3WT were clearly greater than those attached to albumin
serving as a negative control, and were comparable to those of
cells which adhered to fibronectin as shown in FIG. 3. The cell
adhesion and spreading activities of .beta.ig-h3 were
concentration-dependent, as shown in FIGS. 4A and 4B).
.beta.ig-h3.DELTA.RGD lacking the RGD motif was almost equally
effective at supporting cell adhesion and spreading (FIGS. 5A and
5B). These results, taken together, confirm that .beta.ig-h3
supports cell adhesion and spreading, independent of the RGD
motif.
Experimental Example 1
Identification of Cell Surface Receptor of .beta.ig-h3 Involved in
Cell Adhesion Activity of .beta.ig-h3
1-1: Identification of Cell Adhesion Activity Using Matrix Peptide
and Reagent
[0071] In order to identify cell surface receptors involved in the
cell adhesion activity of .beta.ig-h3 protein, an inhibition assay
was performed using various reagents.
[0072] Initially, plastic culture dishes were coated with 10
.mu.g/ml fibronectin, .beta.igh3-WT or .beta.igh3-.DELTA.RGD. HCE
cells were preincubated for 30 min in media containing 5 mM EDTA,
100 .mu.g/ml .beta.igh3-WT, 100 .mu.g/ml igh3-.DELTA.RGD, 1 mM RGD,
1 mM RGE or 100 .mu.g/ml fibronectin, or none of them, and then
assayed for cell adhesion as in Example 1.
[0073] Cell adhesion to .beta.ig-h3 was significantly inhibited by
.beta.ig-h3 itself, RGD peptide and EDTA, and partially inhibited
by fibronectin and EGTA, while being not inhibited by RGE peptide.
Cell adhesion to fibronectin was also significantly inhibited by
fibronectin itself, RGD peptide and EDTA, and partially inhibited
by .beta.ig-h3 and EGTA, but not by RGE peptide, as shown in FIG.
6A. These results indicate that the cell surface receptor for
.beta.ig-h3, which is involved in the cell adhesion activity of
.beta.ig-h3, could be one of the RGD-dependent integrins.
1-2: Effect of Divalent Cations on Cell Adhesion Activity
[0074] To analyze the divalent cation sensitivity of
.beta.ig-h3-mediated adhesion, cells were suspended in
HEPES-buffered saline (HBS) (150 mM NaCl, 25 mM HEPES, 2 mM EDTA,
pH 7.4) at a density of 2.times.10.sup.5 cells/ml and incubated at
37.degree. C. for 30 min. Then, they were washed twice in HBS and
resuspended in the same buffer. Aliquots of cells (50 .mu.l) were
then added to the microculture plate wells and incubated for 30 min
at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 with 50
.mu.l aliquots of HBS containing divalent cations (MnCl.sub.2,
MgCl.sub.2 or CaCl.sub.2) at a concentration twice as large as the
final concentration. Subsequently, they were plated on
ligand-coated dishes to perform the adhesion assay.
[0075] Cell adhesion to .beta.ig-h3 was strongly promoted by
Mn.sup.2+, and to a lesser extent by Mg.sup.2+, but only marginally
by Ca.sup.2+, as shown in FIG. 6B. taken together, the results
demonstrate that the cell surface receptor of .beta.ig-h3 is a kind
of RGD-dependent integrin which requires divalent cations for
interaction with .beta.ig-h3.
1-3: Identification of Cell Surface Receptor of .beta.ig-h3 Using
Monoclonal Antibody Against Integrin
[0076] To identify receptors for .beta.ig-h3, function-blocking
monoclonal antibodies to integrin subunits were examined for their
effect on the adhesion of HCE cells to a surface coated with
.beta.ig-h3. In this regard, initially, HCE (3.times.10.sup.5
cells/ml) were preincubated in an incubation solution in the
presence of each of the monoclonal antibodies (5 .mu.g/ml) against
different types of integrins at 37.degree. C. for 30 min. The
preincubated cells were transferred onto plates precoated with
.beta.ig-h3 proteins and then incubated further at 37.degree. C.
for 1 hour, followed by the quantitative analysis of .beta.ig-h3
binding with hexosaminidase substrate. The values are expressed as
percentages of the number of cells adhering to .beta.ig-h3 in the
absence of monoclonal antibodies.
[0077] Adhesion to the .beta.ig-h3 coated surface was specifically
inhibited by antibody against .alpha.3 subunit. Because the
integrin .alpha.3 subunit is known to couple with the integrin
.beta.1 subunit, anti-.beta.1 antibody significantly blocked cell
adhesion to .beta.ig-h3, as shown in FIG. 6C. Similar results were
observed using HT1080 cells.
[0078] As a control experiment for the function-blocking
antibodies, fibronectin, vitronectin, laminin and type I collagen
were employed as substrata. HCE cells were preincubated with
function-blocking monoclonal antibodies to integrin subunits and
then transferred onto wells coated with 10 .mu.g/ml fibronectin,
vitronectin, type I collagen or laminin. Following incubation, cell
counts of adhered cells were analyzed.
[0079] Cell adhesion to fibronectin was shown to be clearly
inhibited by antibodies to integrins .alpha.3 and .alpha.5.
Adhesion to vitronectin and type I collagen was blocked by
antibodies to integrin .alpha.v and .alpha.2, respectively, whereas
cell adhesion to laminin was inhibited by antibodies to integrins
.alpha.3 and .alpha.6, as shown in FIG. 6D. On the other hand,
antibody to .beta.1 integrin efficiently inhibited cells from
adhering to all ligands mentioned above.
[0080] For another control experiment, K562 cells, known to express
.alpha.5, but not .alpha.3 integrin, were used. K562 cells were
inoculated onto plates coated with .beta.igh3-WT, fibronectin,
laminin, or type I collagen and incubated for 1 hour, followed by
the hexosaminidase analysis. K562 cells did not adhere to
.beta.ig-h3, but adhered to fibronectin and vitronectin. Taken
together, these results suggest integrin .alpha.3.beta.1 is a
specific receptor for .beta.ig-h3 in HCE cells, as shown in FIG.
6E.
Example 2
Identification of Domains Essential to Cell Adhesion Activity of
.beta.ig-h3
[0081] In an attempt to identify essential amino acids conferring
cell adhesion activity of .beta.ig-h3, an examination was made to
determine whether each repeat domain is capable of mediating cell
adhesion.
[0082] Four recombinant proteins corresponding respectively to four
repeat domain were prepared: four .beta.ig-h3 cDNA fragments
encoding amino acids 129-241, 237-377, 368-506, and 498-637,
respectively, were amplified by PCR and cloned into the EcoRV-XhoI
site of pET-29b(+) and the resulting four expression vectors, named
p.beta.ig-h3 D-I, p.beta.ig-h3 D-II, p.beta.ig-h3 D-III, and
p.beta.ig-h3 D-IV, were used to prepare the recombinant proteins,
as shown in FIG. 7. E. coli transformants with the expression
vectors p.beta.ig-h3 D-II and p.beta.ig-h3 D-IV were designated E.
coli BL21/His.beta.-g and E. coli BL21/His.beta.-e and deposited in
the Korean Collection for Type Culture of Korea Research Institute
of Bioscience and Biotechnology (KRIBB) with accession Nos. KCTC
0905BP and KCTC 0904BP, respectively, on Dec. 4, 2000.
[0083] Expression and purification of the recombinant proteins
.beta.ig-h3 D-1, .beta.ig-h3 D-II, .beta.ig-h3 D-III, and
.beta.ig-h3 D-IV followed the procedure described in Example 1-1
and they were identified by SDS-PAGE, as shown in FIG. 8.
[0084] In regard to the mediation of cell adhesion, the 2.sup.nd
and 4.sup.th fas-1 domains were equally active compared to the wild
type .beta.ig-h3 whereas the 1.sup.st fas-1 domain was weak and the
3.sup.rd fas-1 domain was not active at all, as shown in FIG.
9.
[0085] In experiments with function-blocking antibodies to integrin
subunits, both 2.sup.nd and 4.sup.th fas-1 domain-mediated cell
adhesion were almost fully blocked by antibodies to .alpha.3 and
.beta.1 integrins, suggesting that both 2.sup.nd and 4.sup.th fas-1
domains have amino acids essential for interacting with
.alpha.3.beta.1 integrin, as shown in FIG. 10. These results also
support the conclusion that neither H1 nor H2 sequence mediates
cell adhesion activity of .beta.ig-h3 because the 1.sup.st and
3.sup.rd domains are not active in cell adhesion, although they
have H1 or H2 sequence.
Example 3
Identification of Conserved Amino Acid Sequence Essential for Cell
Adhesion of .beta.ig-h3
3-1: Identification of Conserved Motif by Amino Acid Sequence
Alignment
[0086] To find the amino acid sequence responsible for cell
adhesion in 2.sup.nd and 4.sup.th fas-1 domains of .beta.ig-h3,
which independently show cell attachment, a computer search based
on homologies not only among the repeated fas-1 domains of
.beta.ig-h3, but also among fas-1 domains of other proteins was
carried out. As a result, two amino acids, aspartic acid and
isoleucine, near the H2 region, were found to be highly conserved
among various proteins, as shown in FIG. 11. In addition, it was
found that aspartic acid and isoleucine are both conserved in the
2.sup.nd and 4.sup.th fas-1 domains of .beta.ig-h3, which are of
high cell attachment activity, while only aspartic acid is
conserved in the 1.sup.st fas-1 domain, which shows intermediate
cell attachment activity. As for the 3.sup.rd fas-1 domain which
shows no cell attachment activity, it has neither of the two amino
acids. This fact is further evidence that the aspartic acid and
isoleucine residues near the H.sub.2 region are indispensable for
mediating the cell attachment and spreading activity.
3-2: Identification of Cell Adhesion Activity of the Conversed
Amino Acid Sequence Using Substitution Mutants
[0087] To further confirm that the two amino acids are essential
for cell adhesion, the recombinant protein containing the 4.sup.th
fas-1 domain of .beta.ig-h3 was mutated by substitution as shown in
FIG. 12. The substitution mutant of .beta.ig-h3 D-IV was prepared
by PCR and its sequence was confirmed by base sequencing. The
mutant protein was isolated and purified in the same manner as in
Example 1-1 and confirmed on SDS-PAGE, as shown in FIG. 13.
[0088] Examination was made of the cell attachment activity of the
mutated proteins wherein the Pro616, Asp617 and Ile618 of
.beta.igh3 D-IV were, in combination, substituted with Ser, Ala and
Ser, respectively. The mutant protein having Ala instead of Asp617,
named D617A (.beta.igh3 D-IV-PaI) and the mutant protein having Ser
instead of Ile618, named 1618S (.beta.igh3 D-IV-PDs) significantly
blocked cell adhesion whereas the mutant protein having Ser instead
of Pro616, named P616S (.beta.igh3 D-IV-sDI) was found to have no
influence on cell adhesion activity. As for the mutant protein in
which the three amino acids were mutated, named P616S/D617A/1618S
(.beta.igh3 DIV-sas), it also blocked cell adhesion, as shown in
FIG. 14.
[0089] The nearly complete loss of the 1.sup.st fas-1
domain-mediated cell attachment activity in the 1.sup.st fas-1
domain mutated at Asp617 and Ile618 proved that the aspartic acid
at position 617 and isoleucine at position 618 are very important
in mediating the cell attachment activity of .beta.igh3.
Example 4
Identification of .beta.igh3 Domains Effecting Wound Healing
[0090] 4-1: Expression and Purification of Recombinant .beta.igh3
Protein
[0091] To examine whether only the .beta.ig-h3 domains active in
cell adhesion show the same wound healing function as that of the
native .beta.ig-h3 containing all four fas-1 domains, various
recombinant .beta.igh3 proteins were prepared as shown in FIG. 15:
His-.beta.-b containing all of 4 fas-1 domains; .beta.igh3-D-IV
containing the 4.sup.th domain alone; and .beta.igh3-D-IV, 2X, 3X
and 4X, each containing at least one 4.sup.th domain. Showing the
same cell adhesion activity as in .beta.igh3-WT prepared in Example
1, the recombinant .beta.ig-h3 protein His-.beta.-b was prepared
from the recombinant expression vector pET-29.beta. anchoring at
its EcoRV-EcoRI site an Asp718-BglII fragment which was obtained by
deleting a some amino-terminal region from .beta.ig-h3 cDNA. The
recombinant proteins His-.beta.-b and .beta.igh3-D-IV were
expressed and purified in the same manners as in Example 1-1 and
3.
[0092] The recombinant proteins containing at least one 4.sup.th
domain, such as .beta.ig3-D-IV, 2X, 3X and 4X were prepared as
follows. A DNA fragment encoding to amino acid 498-637
corresponding the 4.sup.th domain was obtained by PCR and the PCR
products were blunt-ended by Klenow enzyme. This blunt-ended cDNA
fragment was inserted to the EcoRV site of the p.beta.ig-h3 D-IV,
which contained the 4.sup.th domain of .beta.ig-h3, and the
resulting expression vector was named p.beta.I-h3 D-IV 2X. The
insert of the p.beta.ig-h3 D-IV 2X was excised by digestion with
EcoRV and XhoI and blunt-ended by treatment with Klenow, followed
by inserting the blunt-ended fragment into EcoRV sites of
p.beta.ig-h3 D-IV and p.beta.ig-h3 D-IV 2X. The resulting
expression vectors were named p.beta.ig-h3 D-IV 3X and p.beta.ig-h3
D-IV 4X. Expression of all recombinant proteins was induced for 3
hours in the presence of 1 mM IPTG and isolated by use of Ni-NTA
resin (Qiagen). Isolated recombinant proteins were purified by
elution with 20 mM Tris-HCl comprising 50 mM NaCl and 300 mM
imidazole. .beta.ig-h3 D-IV 2X, 3X and 4X can be produced in large
amounts because they are synthesized as soluble forms, unlike
.beta.ig-h3 recombinant proteins containing all of the four
domains, and do not undergo denaturation, as shown in FIG. 16A.
Electrophoresis using non-denaturing gel revealed that .beta.ig-h3
D-IV did not form polymers while 2X partially formed polymers and
3X and 4X each readily formed polymers, as shown in FIG. 16B.
[0093] E. coli BL21/His .beta.-e4X, which harbors the expression
vector p.beta.ig-h3 D-IV 4X containing four 4.sup.th domains of
.beta.ig-h3, was deposited in the Korean Collection for Type
Culture of Korea Research Institute of Bioscience and Biotechnology
(KRIBB) with accession No. KCTC 0906BP on Dec. 4, 2000.
[0094] Fibronectin, serving as a positive control, was purified
from citrated rat plasma by affinity chromatography using
gelatin-sepharose 4B. The plasma was filtered at room temperature
through non-substituted sepharose 4B and the eluate was loaded onto
gelatin sepharose 4B equilibrated with 0.05 M Tris-Cl containing
0.05 M EACA (.epsilon.-amino caproic acid), 0.02 M sodium citrate
and 0.02% sodium azide. After being eluted, most plasma proteins
were washed with a buffer containing 1 M sodium chloride. Then,
absorbed fibronectin was eluted with 3M uric acid isotonic buffer
which was subsequently dialyzed for about 48 hours against PBS, pH
7.2, to purify fibronectin. Its concentration was determined by UV
absorbance at 280 nm and freeze-dried before being stored at
-20.degree. C.
4-2: Assay for Wound Healing Activity of .beta.ig-h3 D-IV
Containing the 4.sup.th Domain
[0095] To compare wound healing activity between the recombinant
.beta.ig-h3 protein His-.beta.-b, which contains all of the four
fas-1 domains, like native .beta.ig-h3 protein, and the recombinant
.beta.ig-h3 protein .beta.igh3-D-IV, which contains the 4.sup.th
domain alone, ointment bases comprising the recombinant proteins
were tested as follows.
[0096] Four dermal whole layer wounds, each 2 cm in diameter, were
made on the backs of rats and divided into test groups 1-A, 1-B,
1-C and 1-D according to the ointment applied thereto.
[0097] 1-A: coated at a dose of 1 gm per day with an ointment base
combined with no materials.
[0098] 1-B: coated at a dose of 1 gm per day with an ointment in
which fibronectin was combined at a concentration of 100 .mu.g/ml
with a base.
[0099] 1-C: coated at a dose of 1 g per day with an ointment in
which His.beta.-b protein was combined at a concentration of 100
.mu.g/ml with a base.
[0100] 1-D: coated at a dose of 1 g per day with an ointment in
which .beta.igh3-D-IV protein was combined at a concentration of
100 .mu.g/ml with a base.
[0101] The backs of etherized rats were shaved, followed by
sterilizing the shaved region with betadin solution. In test group
1, the back of each rat was cut by use of a No. 15 surgical blade
to form four circular wounds with a diameter of 2 cm penetrating
the whole dermal layers. Ointments for test groups 1-A, 1-B, 1-C
and 1-D were applied at an amount of about 1 g to the wounds which
were then covered with a synthetic dressing (Tegaderm.RTM. 3M) and
lightly bandaged. Application of ointments was performed once every
day.
[0102] With a base of an aqueous material (SamA base), each of the
ointments contained, per 1 g, spermaceti 38 mg, stearyl alcohol 116
mg, polyethylglycol 38 mg, conc. glycerin 192 mg, ethanol 23 mg,
lauryl sodium sulfate mg, ethyl paraoxybenzoate 0.87 mg, butyl
paraoxybenzoate 0.12 mg, and purified water.
[0103] First, morphologies of wounds were observed. The same scale
was positioned near each wound and pictures were taken at the same
distance from each wound. Pictures were scanned in a computer and
used to measure areas of the wounds with the aid of NIH image
analysis system (Bio-Optics). To take the pictures, the muscle was
completely relaxed by etherizing the rats. Measurements were
performed once every other day until the 22.sup.nd day. For
comparing test groups, the measured values were analyzed according
to ANOVA test and Scheffer's test.
[0104] In all test groups, wound areas were observed to be
gradually reduced just after the formation of wounds. The test
groups to which fibronectin and the recombinant .beta.ig-h3 protein
were applied were measured to be more quickly reduced in wound area
than the test group to which the ointment base alone was applied. A
significant difference in wound area was seen after 7 days of
ointment application. Statistically, there were significant
differences (p<0.05) between wounds of group I-A and the other
groups, which did not show a significant difference therebetween.
The results are given in Table 1, below and FIG. 17.
TABLE-US-00001 TABLE 1 Healing Effect of Ointment Bases Combined
with Recombinant .beta.ig-h3 Proteins on Wounds Day Group 0 2* 4 6
8* 10* I-A 3.15 .+-. 0.49 3.09.sup.a .+-. 0.31 2.45 .+-. 0.39 2.17
.+-. 0.46 1.64.sup.a .+-. 0.50 1.80.sup.a .+-. 0.11 (Control) I-B
3.17 .+-. 0.78 2.38.sup.b .+-. 0.55 2.01 .+-. 0.54 1.83 .+-. 0.42
1.39.sup.b .+-. 0.38 1.26.sup.b .+-. 0.18 (Fibronectin) I-C 3.14
.+-. 0.46 2.58.sup.b .+-. 0.47 1.89 .+-. 0.26 1.71 .+-. 0.33
1.42.sup.b .+-. 0.45 1.37.sup.b .+-. 0.71 (His-.beta.-b) I-D 3.15
.+-. 0.43 2.62.sup.b .+-. 0.52 2.37 .+-. 0.45 1.98 .+-. 0.52
1.51.sup.b .+-. 0.21 0.98.sup.b .+-. 0.69 (.beta.ig-h3-D- IV) Day
Group 12 14 16 18 20 22 I-A 0.84 .+-. 0.32 0.56 .+-. 0.31 0.34 .+-.
0.07 0.19 .+-. 0.04 0.17 .+-. 0.05 0.08 .+-. 0.01 (Control) I-B
0.54 .+-. 0.11 0.39 .+-. 0.12 0.25 .+-. 0.12 0.16 .+-. 0.09 0.12
.+-. 0.09 0.06 .+-. 0.03 (Fibronectin) I-C 0.46 .+-. 0.06 0.36 .+-.
0.13 0.26 .+-. 0.09 0.15 .+-. 0.10 0.11 .+-. 0.03 0.03 .+-. 0.01
(His-.beta.-b) I-D 0.44 .+-. 0.24 0.22 .+-. 0.09 0.20 .+-. 0.09
0.18 .+-. 0.10 0.13 .+-. 0.07 0.06 .+-. 0.01 (.beta.ig-h3-D- IV)
Value: mean .+-. SD *p < 0.05 by ANOVA and Scheffe's test
.sup.a,bvertically significant difference of data in statistics
[0105] Histological analysis was conducted under an optical
microscope. Biopsies of wound sites were taken at days 3, 7, 10, 14
and 20, and fixed in 10% formalin and solidified with paraffin. 6
.mu.m slices of the samples were dyed with hematoxylin-eosin
(H&E) and Masson's trichrome before observation under a
microscope. Wound healing effects according to time of each test
material were evaluated through re-epithelialization and formation
of collagenous fibers. In the case of re-epithelialization,
epithelial formation was semi-quantified in such a way that zero
was set for the formation of no epithelial layers, 1+ for
initiation of epithelialization, 2+ for incomplete epithelial layer
structure, and 3+ for complete epithelial layer structure.
Regarding comparison among test groups and differences according to
time within each group, measured values were statistically analyzed
using ANOVA test and Scheffe's test. As for collagenous fiber
formation, it was graded as 1+ for insignificant formation of
collagenous fibers as observed with trichrome dye, 2+ for
scatteringly spaced collagenous fibers, and 3+ for dense
collagenous fibers.
[0106] As observed by an optical microscope, re-epithelialization
appeared to start at day 7 to 10 in test groups 1-B, 1-C and 1-D
and be completed at day 20. In the case of the control group 1-A,
on the other hand, re-epithelialization was not yet initiated even
at day 14 and was not completed at day 20. The results are given in
Table 2, below and FIG. 18.
TABLE-US-00002 TABLE 2 Re-ephithelialization of Wound Day Group 3 7
10 14 20 1-A (Control) 0 0 0 0 2+ 1-B 0 1+ 1+ 2+ 3+ Fibronectin)
1-C (His-.beta.-b) 0 0 1+ 2+ 3+ 1-D (.beta.igD-IV) 0 0 1+ 2+ 3+
[0107] Results for formation of collagenous fibers are given in
Table 3, below. As seen in Table 3, collagenous fibers were not
significantly formed in test groups 1-A and 1-D until day 7 with
maintenance of grade +1, whereas test groups 1-B and 1-C were
graded as 2+. However, all test groups were graded as 2+ at day 10
with relatively rich collagenous fibers. At day 14, it was observed
that collagenous fibers were densely formed and well arranged with
grade +3, as shown in FIG. 19. Naturally, denser collagenous fibers
reflect more improved wound healing progress.
TABLE-US-00003 TABLE 3 Formation Behavior of Collagenous Fibers at
Wound Day Group 3 7 10 14 20 1-A (Control) 1+ 1+ 2+ 3+ 3+ 1-B
(Fibronectin) 1+ 2+ 2+ 3+ 3+ 1-C (His-.beta.-b) 1+ 2+ 2+ 3+ 3+ 1-D
(.beta.igD-IV) 1+ 1+ 2+ 3+ 3+ Formation grade of collagenous fibers
0: negative, 1+: insignificant, 2+: scatteringly formed, 3+: very
dense
4-3: Wound Healing Effect of at Least One 4.sup.th
Domain-Containing Recombinant Proteins .beta.igh3-D-IV, .beta.ig-h3
D-IV 2X, 3X and 4X
[0108] Based on the finding that .beta.igh3-D-IV containing only
the 4.sup.th domain is efficient for wound healing, .beta.igh3-D-IV
2X, 3X and 4X, which contained the 4.sup.th domain in duplicate,
triplicate and quadruplicate, respectively, were prepared in order
to assay for wound healing activity.
[0109] The recombinant proteins were assayed for HCE cell adhesion
activity in the same manner as in Example 1-2. The results are
given in FIG. 20. As seen, the recombinant proteins .beta.ig-h3
D-IV 2X, 3X and 4X were all found to effectively induce the
adhesion of HCE cells.
[0110] In order to examine wound healing effects of the recombinant
proteins, the following experiments were conducted.
[0111] Adult Spraque-Dawley lineage rats with a body weight of
250-300 gm were raised with standard feedstuff at a constant
temperature and humidity.
[0112] In test group 2, four circular dermal whole layer wounds
were made on the back of each rat and coated with chitosan bases
combined with materials of interest:
[0113] 2-A: wound coated with chitosan base only
[0114] 2-B: wound coated with chitosan base in combination with 500
.mu.g/ml of fibronectin
[0115] 2-E: wound coated with chitosan base in combination with 500
.mu.g/ml of .beta.ig-h3 D-IV 3X protein
[0116] 2-F: wound coated with chitosan base in combination with 500
.mu.g/ml of .beta.ig-h3 D-IV 4X protein
[0117] The composites based on chitosan were prepared as follows. 1
g of water soluble chitosan (poly(1-4)
2-amino-2-deoxy-.beta.-D-glucan) with a molecular weight of 600,000
Da was dissolved in 100 ml of sterile distilled water and the
resulting 1% solution was dispensed in aliquot of 2 ml to 12-well
plate (Corning, USA), followed by the addition of 100 .mu.g of
gentamycin per well. Fibronectin, .beta.igh3-D-IV 3X, and
.beta.igh3-D-IV 4X were individually added to a concentration of
500 .mu.g/ml and frozen at -70.degree. C., followed by
freeze-drying in a freeze drier (Ilshin) for 12 hours to give
disc-shaped composites.
[0118] The backs of etherized rats were shaved, followed by
sterilizing the shaved region with betadin solution. Penetrating
the whole dermal layers, four circular wounds with a diameter of 7
mm were formed on the back of each rat. The wounds were covered
with composites used for test groups 2-A, 2-B, 2-E and 2-F,
respectively, and then with Tegaderm.RTM. (3M) and lightly
bandaged. The composites were changed with fresh ones every three
days.
[0119] Wound healing effects were evaluated by determining
appearances of the wounds as in Example 4-2.
[0120] A high wound healing effect was obtained from the composite
containing the recombinant protein .beta.ig-h3 D-IV 3X or 4X.
[0121] All rats, except for all members in the test group 2-A, one
in the test group 2-B and two in test groups 2-E and 2-F each, were
completely recovered from the wound at day 12 to 15. All wound
areas reduced in size just after the formation of wound. As for the
test group 2-A, its wound area was observed to be reduced at a
relatively slow rate throughout the period of healing time. In the
other test groups, the wound areas were reduced greatly in the
first three days, gradually to day 9, and then greatly again.
Turning to comparison among wounds, there were more significant
differences (p<0.05) for the whole period of 15 days in the test
groups 2-B, 2-E and 2-F, than in the test group 2-A, as shown in
Table 4 and FIG. 21.
TABLE-US-00004 TABLE 4 Reduction of Wound Area (mm.sup.2) Day Group
0 3* 6* 9* 12* 15* 2-A (Chitosan) 49.3 .+-. 4.0 34.5 .+-. 0.6.sup.a
26.1 .+-. 0.5.sup.a 13.8 .+-. 0.5.sup.a 10.8 .+-. 0.3.sup.a 3.4
.+-. 0.2.sup.a 2-B 49.2 .+-. 0.5 24.1 .+-. 0.6.sup.b 12.9 .+-.
0.6.sup.b 9.7 .+-. 0.8.sup.b 3.2 .+-. 0.4.sup.b 0.8 .+-. 0.2.sup.b
(Chitosan+Fibronectin) 2-C 49.2 .+-. 1.5 25.3 .+-. 0.7.sup.b 16.6
.+-. 0.6.sup.b 11.2 .+-. 0.5.sup.b 5.0 .+-. 0.8.sup.b 1.2 .+-.
0.2.sup.b (Chitosan+.beta.ig-h3 3X) 2-D 48.5 .+-. 0.4 24.5 .+-.
0.6.sup.b 14.1 .+-. 0.7.sup.b 9.6 .+-. 0.6.sup.b 4.2 .+-. 0.3.sup.b
1.1 .+-. 0.2.sup.b (Chitosan+.beta.ig-h3 4X) Value: Mean .+-. S.D
.sup.+: p < 0.05 by ANOVA test .sup.a,bsignificant difference of
data in statistics
[0122] Consequently, the recombinant proteins of the present
invention, which contain the 2.sup.nd and 4.sup.th domains of
.beta.ig-h3, alone or incombination, or in multiplicate are
effective for cell adhesion and wound healing and ultimately can be
utilized in developing cell culture and wound healing agents.
INDUSTRIAL APPLICABILITY
[0123] In the present invention, there are provided recombinant
proteins containing at least one of the 2.sup.nd and 4.sup.th
domains of .beta.ig-h3 in which one aspartic acid and one
isoleucine residue, known to be essential for association with
integrin, are highly conserved. Also, the recombinant proteins
themselves are useful for cell adhesion and wound healing, making a
contribution to the development of cell culture methods and wound
healing agents.
[0124] The present invention has been described in an illustrative
manner, and it is to be understood that the terminology used is
intended to be in the nature of description rather than of
limitation. Many modifications and variations of the present
invention are possible in light of the above teachings. Therefore,
it is to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described.
Sequence CWU 1
1
27126PRTHUMAN 1Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His His
Ala Thr Asn1 5 10 15Gly Val Val His Leu Ile Asp Lys Val Ile 20
25226PRTPIG 2Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His His
Ala Thr Asn1 5 10 15Gly Val Val His Leu Ile Asp Lys Val Ile 20
25326PRTCHICKEN 3Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His
His Ala Thr Asn1 5 10 15Gly Val Val His Val Ile Asp Lys Val Ile 20
25427PRTHUMAN 4Ile Asn Gly Lys Ala Ile Ile Ser Asn Lys Asp Ile Leu
Ala Thr Asn 1 5 10 15Gly Val Ile His Tyr Ile Asp Glu Leu Leu Ile 20
25527PRTPIG 5Ile Asn Gly Lys Pro Ile Ile Ser Asn Lys Asp Val Leu
Ala Thr Asn1 5 10 15Gly Val Ile His Phe Ile Asp Glu Leu Leu Ile 20
25627PRTCHICKEN 6Leu Asn Gly Arg Ala Ile Ile Ala Asn Lys Asp Ile
Leu Ala Thr Asn1 5 10 15Gly Val Val His Gly Val Asn Glu Leu Leu Ile
20 25727PRTHUMAN 7Val Asn Gly Ile Lys Met Val Asn Lys Lys Asp Ile
Val Thr Asn Asn1 5 10 15Gly Val Ile His Leu Ile Asp Gln Val Leu Ile
20 25827PRTMOUSE 8Ile Asn Gly Ile Lys Met Val Asn Lys Lys Asp Ile
Val Thr Lys Asn1 5 10 15Gly Val Ile His Leu Ile Asp Glu Val Leu Ile
20 25925PRTHUMAN 9Val Asn Lys Glu Pro Val Ala Glu Pro Asp Ile Met
Ala Thr Asn Gly1 5 10 15Val Val His Val Ile Thr Asn Val Leu 20
251025PRTPIG 10Val Asn Lys Glu Pro Val Ala Glu Ala Asp Ile Met Ala
Thr Asn Gly1 5 10 15Val Val His Thr Ile Asn Thr Val Leu 20
251125PRTCHICKEN 11Val Asn Lys Glu Pro Val Ala Glu Ser Asp Ile Met
Ala Thr Asn Gly1 5 10 15Val Ile His Ala Val Ser Ser Val Leu 20
251228PRTSLL1735 Homolog 12Val Lys Asn Ala Thr Val Leu Ala Ala Asp
Ile Glu Ala Asp Asn Gly1 5 10 15Ile Ile His Val Ile Asp Asn Val Ile
Leu Met Gly 20 251328PRTSynechocystis PCC7002 13Val Lys Asn Ala Thr
Val Ile Ile Pro Asp Ile Glu Ala Asp Asn Gly1 5 10 15Ile Ile His Val
Ile Asp Asn Val Ile Leu Met Gly 20 251426PRTSynechocystis PCC6803
14Val Asn Lys Ala Thr Val Ile Ser Ala Asp Val Asp Ala Ser Asn Gly1
5 10 15Val Ile His Val Ile Asp Gln Val Ile Leu 20 251525PRTHUMAN
15Val Asn Glu Leu Lys Ser Lys Glu Ser Asp Ile Met Thr Thr Asn Gly1
5 10 15Val Ile His Val Val Asp Lys Leu Leu 20 251625PRTMOUSE 16Val
Asn Glu Leu Lys Ser Lys Glu Ser Asp Ile Met Thr Thr Asn Gly1 5 10
15Val Ile His Val Val Asp Lys Leu Leu 20 251726PRTDrosophila
melanogaster 17Ile Asn Asn Leu Ala Lys Ile Ile Asp Ala Asp Ile Met
Gly Thr Asn1 5 10 15Gly Val Leu His Val Ile Asp Thr Ile Leu 20
251826PRTEchinoidea 18Ser Lys Ala Ser Arg Val Ile Leu Arg Asp Ile
Pro Thr Thr Asn Gly1 5 10 15Val Ile Gln Val Ile Asp Arg Val Ile Leu
20 251927PRTDrosophila melanogaster 19Lys Ile Glu Asn Ala Gly Val
Thr Lys Cys Asp Val Val Ala Thr Asn1 5 10 15Gly Ile Leu His Glu Ile
Asn Asp Ile Ile Val 20 252026PRTEchinoidea 20Thr Ala Asn Gly Ala
Arg Val Val Glu Ala Asp Arg Lys Ala Ser Ser1 5 10 15Gly Leu Ile His
Val Val Asp Lys Val Ile 20 252129PRTArtificial Sequenceconsensus
21Val Asn Asn Ala Ala Arg Val Val Lys Ala Asp Ile His Ala Thr Asn1
5 10 15Gly Val Ile His Val Ile Asp Lys Val Leu Ile Met Gly 20
2522683PRTHuman 22Met Ala Leu Phe Val Arg Leu Leu Ala Leu Ala Leu
Ala Leu Ala Leu1 5 10 15Gly Pro Ala Ala Thr Leu Ala Gly Pro Ala Lys
Ser Pro Tyr Gln Leu 20 25 30Val Leu Gln His Ser Arg Leu Arg Gly Arg
Gln His Gly Pro Asn Val 35 40 45Cys Ala Val Gln Lys Val Ile Gly Thr
Asn Arg Lys Tyr Phe Thr Asn 50 55 60Cys Lys Gln Trp Tyr Gln Arg Lys
Ile Cys Gly Lys Ser Thr Val Ile65 70 75 80Ser Tyr Glu Cys Cys Pro
Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly 85 90 95Cys Pro Ala Ala Leu
Pro Leu Ser Asn Leu Tyr Glu Thr Leu Gly Val 100 105 110Val Gly Ser
Thr Thr Thr Gln Leu Tyr Thr Asp Arg Thr Glu Lys Leu 115 120 125Arg
Pro Glu Met Glu Gly Pro Gly Ser Phe Thr Ile Phe Ala Pro Ser 130 135
140Asn Glu Ala Trp Ala Ser Leu Pro Ala Glu Val Leu Asp Ser Leu
Val145 150 155 160Ser Asn Val Asn Ile Glu Leu Leu Asn Ala Leu Arg
Tyr His Met Val 165 170 175Gly Arg Arg Val Leu Thr Asp Glu Leu Lys
His Gly Met Thr Leu Thr 180 185 190Ser Met Tyr Gln Asn Ser Asn Ile
Gln Ile His His Tyr Pro Asn Gly 195 200 205Ile Val Thr Val Asn Cys
Ala Arg Leu Leu Lys Ala Asp His His Ala 210 215 220Thr Asn Gly Val
Val His Leu Ile Asp Lys Val Ile Ser Thr Ile Thr225 230 235 240Asn
Asn Ile Gln Gln Ile Ile Glu Ile Glu Asp Thr Phe Glu Thr Leu 245 250
255Arg Ala Ala Val Ala Ala Ser Gly Leu Asn Thr Met Leu Glu Gly Asn
260 265 270Gly Gln Tyr Thr Leu Leu Ala Pro Thr Asn Glu Ala Phe Glu
Lys Ile 275 280 285Pro Ser Glu Thr Leu Asn Arg Ile Leu Gly Asp Pro
Glu Ala Leu Arg 290 295 300Asp Leu Leu Asn Asn His Ile Leu Lys Ser
Ala Met Cys Ala Glu Ala305 310 315 320Ile Val Ala Gly Leu Ser Val
Glu Thr Leu Glu Gly Thr Thr Leu Glu 325 330 335Val Gly Cys Ser Gly
Asp Met Leu Thr Ile Asn Gly Lys Ala Ile Ile 340 345 350Ser Asn Lys
Asp Ile Leu Ala Thr Asn Gly Val Ile His Tyr Ile Asp 355 360 365Glu
Leu Leu Ile Pro Asp Ser Ala Lys Thr Leu Phe Glu Leu Ala Ala 370 375
380Glu Ser Asp Val Ser Thr Ala Ile Asp Leu Phe Arg Gln Ala Gly
Leu385 390 395 400Gly Asn His Leu Ser Gly Ser Glu Arg Leu Thr Leu
Leu Ala Pro Leu 405 410 415Asn Ser Val Phe Lys Asp Gly Thr Pro Pro
Ile Asp Ala His Thr Arg 420 425 430Asn Leu Leu Arg Asn His Ile Ile
Lys Asp Gln Leu Ala Ser Lys Tyr 435 440 445Leu Tyr His Gly Gln Thr
Leu Glu Thr Leu Gly Gly Lys Lys Leu Arg 450 455 460Val Phe Val Tyr
Arg Asn Ser Leu Cys Ile Glu Asn Ser Cys Ile Ala465 470 475 480Ala
His Asp Lys Arg Gly Arg Tyr Gly Thr Leu Phe Thr Met Asp Arg 485 490
495Val Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp
500 505 510Asn Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly
Leu Thr 515 520 525Glu Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe
Ala Pro Thr Asn 530 535 540Glu Ala Phe Arg Ala Leu Pro Pro Arg Glu
Arg Ser Arg Leu Leu Gly545 550 555 560Asp Ala Lys Glu Leu Ala Asn
Ile Leu Lys Tyr His Ile Gly Asp Glu 565 570 575Ile Leu Val Ser Gly
Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu 580 585 590Gln Gly Asp
Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val 595 600 605Asn
Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val 610 615
620Val His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn Arg Pro
Gln625 630 635 640Glu Arg Gly Asp Glu Leu Ala Asp Ser Ala Leu Glu
Ile Phe Lys Gln 645 650 655Ala Ser Ala Phe Ser Arg Ala Ser Gln Arg
Ser Val Arg Leu Ala Pro 660 665 670Val Tyr Gln Lys Leu Leu Glu Arg
Met Lys His 675 68023113PRTArtificial Sequence1st fas-1 domain of
betaIG-h3 23Arg Pro Glu Met Glu Gly Pro Gly Ser Phe Thr Ile Phe Ala
Pro Ser1 5 10 15Asn Glu Ala Trp Ala Ser Leu Pro Ala Glu Val Leu Asp
Ser Leu Val 20 25 30Ser Asn Val Asn Ile Glu Leu Leu Asn Ala Leu Arg
Tyr His Met Val 35 40 45Gly Arg Arg Val Leu Thr Asp Glu Leu Lys His
Gly Met Thr Leu Thr 50 55 60Ser Met Tyr Gln Asn Ser Asn Ile Gln Ile
His His Tyr Pro Asn Gly65 70 75 80Ile Val Thr Val Asn Cys Ala Arg
Leu Leu Lys Ala Asp His His Ala 85 90 95Thr Asn Gly Val Val His Leu
Ile Asp Lys Val Ile Ser Thr Ile Thr 100 105
110Asn24141PRTArtificial Sequence2nd fas-1 domain of betaIG-h3
24Ser Thr Ile Thr Asn Asn Ile Gln Gln Ile Ile Glu Ile Glu Asp Thr1
5 10 15Phe Glu Thr Leu Arg Ala Ala Val Ala Ala Ser Gly Leu Asn Thr
Met 20 25 30Leu Glu Gly Asn Gly Gln Tyr Thr Leu Leu Ala Pro Thr Asn
Glu Ala 35 40 45Phe Glu Lys Ile Pro Ser Glu Thr Leu Asn Arg Ile Leu
Gly Asp Pro 50 55 60Glu Ala Leu Arg Asp Leu Leu Asn Asn His Ile Leu
Lys Ser Ala Met65 70 75 80Cys Ala Glu Ala Ile Val Ala Gly Leu Ser
Val Glu Thr Leu Glu Gly 85 90 95Thr Thr Leu Glu Val Gly Cys Ser Gly
Asp Met Leu Thr Ile Asn Gly 100 105 110Lys Ala Ile Ile Ser Asn Lys
Asp Ile Leu Ala Thr Asn Gly Val Ile 115 120 125His Tyr Ile Asp Glu
Leu Leu Ile Pro Asp Ser Ala Lys 130 135 14025139PRTArtificial
Sequence3rd fas-1 domain of betaIG-h3 25Asp Glu Leu Leu Ile Pro Asp
Ser Ala Lys Thr Leu Phe Glu Leu Ala1 5 10 15Ala Glu Ser Asp Val Ser
Thr Ala Ile Asp Leu Phe Arg Gln Ala Gly 20 25 30Leu Gly Asn His Leu
Ser Gly Ser Glu Arg Leu Thr Leu Leu Ala Pro 35 40 45Leu Asn Ser Val
Phe Lys Asp Gly Thr Pro Pro Ile Asp Ala His Thr 50 55 60Arg Asn Leu
Leu Arg Asn His Ile Ile Lys Asp Gln Leu Ala Ser Lys65 70 75 80Tyr
Leu Tyr His Gly Gln Thr Leu Glu Thr Leu Gly Gly Lys Lys Leu 85 90
95Arg Val Phe Val Tyr Arg Asn Ser Leu Cys Ile Glu Asn Ser Cys Ile
100 105 110Ala Ala His Asp Lys Arg Gly Arg Tyr Gly Thr Leu Phe Thr
Met Asp 115 120 125Arg Val Leu Thr Pro Pro Met Gly Thr Val Met 130
13526140PRTArtificial Sequence4th fas-1 domain of betaIG-h3 26Leu
Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn1 5 10
15Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu
20 25 30Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn
Glu 35 40 45Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu
Gly Asp 50 55 60Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly
Asp Glu Ile65 70 75 80Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg
Leu Lys Ser Leu Gln 85 90 95Gly Asp Lys Leu Glu Val Ser Leu Lys Asn
Asn Val Val Ser Val Asn 100 105 110Lys Glu Pro Val Ala Glu Pro Asp
Ile Met Ala Thr Asn Gly Val Val 115 120 125His Val Ile Thr Asn Val
Leu Gln Pro Pro Ala Asn 130 135 14027137PRTArtificial Sequence4th
fas-1 domain of betaIG-h3 27Pro Met Gly Thr Val Met Asp Val Leu Lys
Gly Asp Asn Arg Phe Ser1 5 10 15Met Leu Val Ala Ala Ile Gln Ser Ala
Gly Leu Thr Glu Thr Leu Asn 20 25 30Arg Glu Gly Val Tyr Thr Val Phe
Ala Pro Thr Asn Glu Ala Phe Arg 35 40 45Ala Leu Pro Pro Arg Glu Arg
Ser Arg Leu Leu Gly Asp Ala Lys Glu 50 55 60Leu Ala Asn Ile Leu Lys
Tyr His Ile Gly Asp Glu Ile Leu Val Ser65 70 75 80Gly Gly Ile Gly
Ala Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys 85 90 95Leu Glu Val
Ser Leu Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro 100 105 110Val
Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val His Val Ile 115 120
125Thr Asn Val Leu Gln Pro Pro Ala Asn 130 135
* * * * *