U.S. patent application number 11/731464 was filed with the patent office on 2007-09-20 for decomposition and modification of silicate and silicone by silicase and use of the reversible enzyme.
Invention is credited to Anatoli Krasko, Werner E.G. Muller, Heinz Schroder.
Application Number | 20070218044 11/731464 |
Document ID | / |
Family ID | 32010159 |
Filed Date | 2007-09-20 |
United States Patent
Application |
20070218044 |
Kind Code |
A1 |
Muller; Werner E.G. ; et
al. |
September 20, 2007 |
Decomposition and modification of silicate and silicone by silicase
and use of the reversible enzyme
Abstract
Silicatein is an enzyme of silicate-forming organisms used for
the synthesis of their silicate scaffold. The present invention
relates to the use of highly-expressed and highly active
recombinant silicatein, silicatein isolated from natural sources
after gene induction as well as silicatein-fusion proteins for the
synthesis of amorphous silicon dioxide (silicic acids and
silicates), siloxanes as well as modification of these compounds
and their technical use.
Inventors: |
Muller; Werner E.G.;
(Wiesbaden, DE) ; Schroder; Heinz; (Wiesbaden,
DE) ; Krasko; Anatoli; (Mainz, DE) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
32010159 |
Appl. No.: |
11/731464 |
Filed: |
March 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10530240 |
Jul 27, 2005 |
7229807 |
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PCT/EP03/10983 |
Oct 2, 2003 |
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11731464 |
Mar 29, 2007 |
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Current U.S.
Class: |
424/94.1 ;
423/325; 435/168; 435/183; 435/243; 435/320.1; 435/4; 536/23.1 |
Current CPC
Class: |
C12N 9/88 20130101; C12P
3/00 20130101 |
Class at
Publication: |
424/094.1 ;
423/325; 435/168; 435/183; 435/243; 435/320.1; 435/004;
536/023.1 |
International
Class: |
A61K 38/43 20060101
A61K038/43; A61K 31/00 20060101 A61K031/00; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12N 9/00 20060101 C12N009/00; C12P 3/00 20060101
C12P003/00; C12Q 1/00 20060101 C12Q001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 2002 |
DE |
102 46 186.4 |
Claims
1. Method for the in vitro or in vivo degradation of amorphous or
crystalline silicone dioxide (condensation products of the silicic
acid, silicates), silicones and other silicon (IV)- or metal
(IV)-compounds as well as of mixed polymers of these compounds,
wherein a polypeptide or a metal complex of a polypeptide is used
for the degradation, characterized in that the polypeptide
comprises an animal, bacterial, plant or fungal carbonic anhydrase
domain that exhibits a sequence similarity of at least 25% to the
sequence shown in SEQ ID No. 1.
2. Method for the synthesis of amorphous silicone dioxide
(condensation products of the silicic acid, silicates), silicones
and other silicon (IV)- or metal (IV)-compounds as well as of mixed
polymers of these compounds, wherein a polypeptide or a metal
complex of a polypeptide is used for the synthesis, characterized
in that the polypeptide comprises an animal, bacterial, plant or
fungal carbonic anhydrase domain that exhibits a sequence
similarity of at least 25% to the sequence shown in SEQ ID No.
1.
3. Method according to claim 2, characterized in that compounds
such as silicic acids, monoalkoxysilantrioles, dialkoxysilandioles,
trialkoxysilanoles, tetraalkoxysilanes, alkyl- or
aryl-silantrioles, alkyl- or aryl-monoalkoxysilandioles, alkyl- or
aryl-dialkoxysilanoles, alkyl- or aryl-trialkoxysilanes or other
metal(IV)-compounds are used as reactants (substrates) for the
synthesis.
4. Method according to claim 3, wherein mixed polymers having a
defined composition are produced by using defined mixtures of the
compounds.
5. Method according to any of claims 2 to 4, wherein the formation
of defined two- and three-dimensional structures occurs by the
polypeptide or a metal complex of the polypeptide or the binding of
the polypeptide or a metal complexes of the polypeptide to other
molecules or the surfaces of glass, metals, metal oxides, plastics,
biopolymers or other materials as a template.
6. Method for the modification of a silicic acid or silicon(IV)- or
metal (IV)-compound containing structure or surface, wherein a
polypeptide or a metal complex of a polypeptide is used for the
modification, characterized in that the polypeptide comprises an
animal, bacterial, plant or fungal carbonic anhydrase domain that
exhibits a sequence similarity of at least 25% to the sequence
shown in SEQ ID No. 1.
7. Method according to claim 6, wherein the silicic acid-containing
structure or surface is present in form of a precious stone or
semi-precious stone.
8. Method according to claim 6 or 7, wherein the modification
comprises a smoothing, an etching or the production of burrows of
the silicic acid or silicon(IV)- or metal(IV)-compound-containing
structure or surface by the polypeptide or a metal complex of the
polypeptide.
9. Chemical compound or silicic acid-containing structure or
surface, obtained according to a method of the preceding
claims.
10. Silicic acid-containing structure or surface according to claim
9 in the form of a precious stone or semi-precious stone.
11. Polypeptide of a silicase from Suberites domuncula according to
SEQ ID Nr. 1 or a polypeptide being homologous thereto, which in
the amino acid sequence of the carbonic anhydrase domain exhibits a
sequence similarity of at least 25% to the sequence shown in SEQ ID
No. 1, a metal complex of the polypeptide, or parts thereof.
12. Nucleic acid, in particular according to SEQ ID No. 2,
characterized in that it essentially encodes for a polypeptide
according to claim 11.
13. Nucleic acid according to claim 12, characterized in that it is
present in the form of a DNA, cDNA, RNA or mixtures thereof.
14. Nucleic acid according to claim 12 or 13, characterized in that
the sequence of the nucleic acid has at least one intron and/or a
polyA-sequence.
15. Nucleic acid according to any of claims 12 to 14 in the form of
its complementary "antisense"-sequence.
16. Nucleic acid according to any of claims 12 to 15 in the form of
a (a) fusion protein-(chimeric protein) construct, (b) construct
having a separate protein-expression (protease-cleavage site) or
(c) construct having a separate protein-expression
(cassette-expression).
17. Nucleic acid according to any of claims 12 to 16, characterized
in that the nucleic acid has been synthetically produced.
18. Vector, preferably in the form of a plasmid, shuttle vector,
phagemid, cosmid, expression vector, retroviral vector, adenoviral
vector or particle, nanoparticle or liposome, comprising a nucleic
acid according to any of claims 12 to 17.
19. Vector, preferably in the form of a nanoparticle or liposome,
comprising a polypeptide according to claim 11.
20. Host cell, transfected with a vector or infected or transduced
with a particle according to claim 18 or 19.
21. Host cell according to claim 20, characterized in that it
expresses a polypeptide according to claim 1, a metal complex of
the polypeptide or parts thereof.
22. Polypeptide according to claim 11, characterized in that the
polypeptide has been synthetically produced.
23. Polypeptide according to claim 11, characterized in that the
polypeptide or the metal complex of the polypeptide is present in a
prokaryotic or eukaryotic cell extract or lysate.
24. Polypeptide according to claim 23, characterized in that the
polypeptide or the metal complex of the polypeptide is present
being purified essentially free of other proteins.
25. Method for identifying of inhibitors or activators of a
polypeptide of a silicase from Suberites domuncula according to SEQ
ID No. 1 or a polypeptide being homologous thereto that in the
amino acid sequence of the carbonic anhydrase domain has at least
25% sequence similarity to the sequence shown in SEQ ID No. 1,
wherein a) a polypeptide of a silicase from Suberites domuncula
according to SEQ ID No. 1 or a polypeptide being homologous thereto
that in the amino acid sequence of the carbonic anhydrase domain
has at least 25% sequence similarity to the sequence shown in SEQ
ID No. 1 is provided, b) the polypeptide from step a) is contacted
with a potential inhibitor or activator, and c) the ability of the
polypeptide is measured to degrade or synthesize silicate or
silicones.
26. Method according to claim 25, wherein the polypeptide of a
silicase from Suberites domuncula according to SEQ ID No. 1 or a
polypeptide being homologous thereto that in the amino acid
sequence of the carbonic anhydrase domain has at least 25% sequence
similarity to the sequence shown in SEQ ID No. 1 is provided in
vivo, in a cellular extract or lysate or in purified form.
27. Method for producing a pharmaceutical composition, comprising
a) identifying of an inhibitor or activator according to claim 25
or 26 and b) mixing of the identified inhibitor or activator with a
pharmaceutically acceptable carrier or excipient.
28. Use of a polypeptide or a nucleic acid or pharmaceutical
composition according to any of the preceding claims for the
prevention or therapy of silicosis.
29. Use according to claim 28, wherein the prevention and therapy
of silicosis occurs by dissolving of quartz crystals
30. Use of a polypeptide or a nucleic acid or pharmaceutical
composition according to any of the preceding claims for the
resorption or for modulating the resorbability of silicones and
silicone implants.
31. Use of a nucleic acid according to any of the preceding claims
for transfecting cells for the resorption or for modulating the
resorbability of silicones and silicone implants.
Description
1. STATE OF THE ART
[0001] Silicon is the second-most element of the earth's crust and
is present in all kinds of different compounds. Silicon compounds
do not only represent most of the species of this class of
minerals, but are also very important from an economical point of
view. Technically used materials that are composed of silicates
are, for example, glass, porcelain, enamel, clay products, cement
and water glass. Some silicates exhibit catalytic properties. Their
diversity in structures and the technical uses are further
expanded, if other elements, in particular aluminum, occupy some of
the lattice positions that are otherwise occupied by silicon. Thus,
the alumo silicates, belonging to which are feldspars and
zeolithes, have importance due to, amongst others, their molecular
sieve and ions exchange properties. Other silicon-compounds such as
the silicones (siloxanes), amongst others, also are of medical
importance, such as for the production of implants.
1.1. Silicon Dioxide
[0002] Silicon dioxide (SiO.sub.2) can be found both in
crystallized and amorphous form. Quartz, tridymite, and
cristobalite, amongst others, belong to the different forms of
crystalline SiO.sub.2. Achat, opal, and flint stone represent
amorphous silicon dioxide-materials. In all these silicon has the
coordination-number 4 and is tetraedrically surrounded by four
oxygen-atoms.
[0003] Furthermore, the shells of diatoms (diatomeae) and the
needles (spicules) of diatomeous sponges consist out of amorphous
SiO.sub.2.
1.2. Silicic Acids and Silicates
[0004] The tetraedrically-built [SiO.sub.2].sup.4-ion tends to
polymerization by linking SiO.sub.4-Units, wherein in each case two
Si-atoms are linked together by an O-atom. In this, at first
ortho-disilicic acid (pyro-silicic acid; H.sub.6Si.sub.2O.sub.7) is
formed from ortho-silicic acid by condensation (splitting off
water). The further condensation via the poly-silicic acids leads
to the meta-silicic acids [(H.sub.2SiO.sub.3)].sub.n. In case of
smaller numbers of SiO.sub.4-units (n=3, 4 or 6) also ring-shaped
molecules can be formed through this.
[0005] The salts of the silicic acids, the alkali silicates, which,
for example, can be obtained by melting of quartz with soda, brine
or potassium carbonate, in addition to [SiO.sub.4].sup.4- anions,
also contain [Si.sub.2O.sub.7].sup.6- and [Si.sub.3O.sub.10].sup.8-
anions, and larger anions. Such ortho-disilicic acids
(ortho-silicates), having the structure Me.sub.2SiO.sub.4, contain
single [SiO.sub.4].sup.4- anions. After acidification of such an
alkali silicate-solution, the acid molecules that are formed by the
uptake of protons, condensate with each other to form poly-silicic
acids, whereby the solution becomes gel-like. Upon further progress
of the condensation, three-dimensional structures are formed from
the chains or nets that are first obtained, that correspond to the
composition SiO.sub.2.
[0006] The silicates can be classified into: 1.) Silicates with
discrete anions, namely 1a) island-silicates (ortho-silicates
having the anion [SiO.sub.4].sup.2-; example: phenacite, olivine,
zirconium), 1b) Group-silicates (Linkage of the
SiO.sub.4-tetraeders to form short chain units: example:
di-silicates and tri-silicates) and 1c) Ring-silicates (the
SiO.sub.4-tetraeders are arranged in ring form, example: benitoid
with 3-ring, axinite with 4-ring, and beryllium with 6-ring), 2.
Chain-silicates and ribbon-silicates (chain-like
SiO.sub.4-tetraeders are bound to each other; representing polymers
of the anions [SiO.sub.3].sup.2-, and ribbon-like molecules that
are formed by linking several SiO.sub.4-chains; examples:
homblende, asbestos). 3. Layer or sheet-silicate (made from even
layers of tetraeders that represent polymers of the anions
[Si.sub.4O.sub.10].sup.4- and are held together by cations stored
in-between; examples: talcum, caolinit, and 4. Scaffold-silicates
(linkage of the tetraedic SiO.sub.4-groups into three-dimensional
lattices; example: different modifications of silicon dioxide, such
as feldspatuses).
[0007] General literature: Hinz, Silicat-Lexikon (2 Bd.), Berlin:
Akademie Verl. 1985; Liebau, Structural Chemistry of Silicates,
Berlin: Springer 1985; Petzold and Hinz, Einfuhrung in die
Grundlagen der Silicatchemie, Stuttgart: Enke 1979; CD Rompp Chemie
Lexikon-Version 1.0, Stuttgart/New York: Georg Thieme Verlag
1995.
1.3. Silicones
[0008] Different silicones (siloxanes) are generated by a partial
replacement of the OH-group in the silicic acid by single-bond
organylic residues that do not participate in the condensation
process. They are classified into: 1.) linear polysiloxanes
(construction type: R.sub.3SiO[R.sub.2SiO].sub.nSiR.sub.3), 2)
branched polysiloxanes (with tri-functional or tetra-functional
siloxane-units at their branching sites), 3) cyclic polysiloxanes
(from di-functional siloxane-units) and 4) cross linked polymers
(chain- or ring-form molecules are linked into two- or
three-dimensional networks).
[0009] Silicones are important technical materials. The viscosity
of the high molecular weight silicones (silicone oils) consisting
of chain-macromolecules, increases with increasing chain length.
Silicones that are cross-linked to a low extent exhibit
rubber-elasticity (silicone rubber), highly cross-linked chains are
resin-like (silicone resins).
1.4. Silicatein
[0010] Some of the above-mentioned silicon compounds can only be
produced in a cost-intensive manner or are present only in small
amounts as mineral resources, respectively, and can therefore only
be isolated with considerable effort. The process of the chemical
synthesis of silicates requires drastic conditions, such as high
pressure and high temperature.
[0011] In contrast, with the aid of specific enzymes organisms (in
particular sponges and algae) are able to form silicate scaffolds
under natural conditions, i.e. at low temperature and low pressure.
The advantages of this pathway are: high specificity, coordinated
formation, adjustability, and the possibility for synthesizing
nanostructures.
[0012] The isolation and purification of a silicate-forming enzyme
(silicatein) was recently described for the first time: Shimizu,
K., et al., Proc. Natl. Acad. Sci. USA 95: 6234-6238 (1998).
[0013] Nevertheless, this results in the problem that the isolation
and the purification of the enzyme (silicatein) is time-consuming
and laborious, and that only relatively low amounts can be
achieved.
[0014] One possible approach is the synthesis of the recombinant
protein (recombinant silicatein) with the aid of the known cDNA- or
gene-sequence. This allows for the effective enzymatic synthesis of
silicates.
[0015] In case of the production of the recombinant silicateins
from the sponges Suberites domuncula and Tethya aurantia, the
problem occurred that by using the methods according to the state
of the art only very low yields could be achieved and that the
recombinant protein exhibited only low enzymatic activity. The
present invention describes that, by specific modification of the
expression conditions, recombinant silicatein can be produced in
high yields and with high specific activity. Furthermore, the
modified recombinant enzyme exhibits a higher pH and temperature
stability than the natural one and the recombinant one having a
complete cDNA-sequence. The modified recombinant protein
furthermore exhibits an enzymatic activity over a broad pH
(4.5-10), in contrast to the natural and recombinant protein with
complete cDNA-sequence that is active at pH-values in the neutral
range (pH 7.0).
[0016] By way of production of a specific polyclonal antibody and
subsequent coupling to a solid phase, a fast and effective
affinity-chromatography purification of the enzyme can be
achieved.
[0017] The use of fusion proteins and the application of different
starting substrates lead to numerous possibilities for variations
and technical applications.
1.4. Biomineralisation (Formation of Biogenic Silicon dioxide) in
Siliceous Sponges
[0018] Many silicon compounds can only be produced in a
cost-intensive manner, The process of the chemical synthesis of the
silicates often requires drastic conditions, such as high pressure
and high temperature. In contrast, siliceous sponges--in addition
to diatoms--are able to form silicate scaffolds under mild
conditions with the aid of specific enzymes, i.e. at relatively low
temperature and low pressure. Furthermore, in these organisms the
SiO2-synthesis is characterized by a high specificity,
controllability and the possibility of the synthesis of defined
microstructures (nanostructures)
[0019] The main elements of the skeleton of the siliceous sponges
are the needle-like spicules that in the group of the demospongiae
(horn sponges) and hexactinellidae (glass sponges) consist out of
amorphous non-crystalline silicon dioxide. The demospongiae and
hexactinellidae are the only metazoes that have silicon dioxide
instead of calcium in their skeleton.
[0020] The opaque silicon dioxide in the spicules of the siliceous
sponges contains 6-13% water resulting in the approximate formula
(SiO.sub.2).sub.25H.sub.2O (Schwab D W, Shore R E (1971) Mechanism
of internal stratification of siliceous spicules. Nature 232:
501-502).
[0021] An enzyme that is involved in the synthesis of the
SiO.sub.2-skeletton in silicate forming organisms, and its
technical use was described (PCT/US99/30601. Methods, compositions,
and biomimetic catalysts, such as silicateins and block
copolypeptides, used to catalyze and spatially direct the
polycondensation of silicon alkoxide, metal alkoxide, and their
organic conjugates to make silica, polysiloxanes,
polymetallo-oxanes, and mixed poly (silicon/metallo) oxane
materials under environmentally benign conditions.
Inventors/applicants: Morse D E, Stucky G D, Deming, T D, Cha J,
Shimizu K, Zhou Y; DE 10037270 A 1. Silicatein-vermittelte
Synthesis von amorphen Silicaten and Siloxane and ihre Verwendung.
Deutsches Patentamt 2000. Applicant and inventor: Muller W E G,
Lorenz B, Krasko A, Schroder HC; PCT/EP01/08423.
Silicatein-mediated synthesis of amorphous silicates and siloxane
and use thereof. Inventors/Applicants: Muller W E G, Lorenz B,
Krasko A, Schroder H C). This enzyme was cloned from the marine
siliceous sponge Suberites domuncula (Krasko A, Batel R, Schroder H
C, M{umlaut over (ue)}ller I M, Muller W E G (2000) Expression of
silicatein and collagen genes in the marine sponge S. domuncula is
controlled by silicate and myotrophin. Europ J Biochem 267:
4878-4887). The enzyme being isolated from natural sources
("silicatein") is able to synthesize amorphous silicon dioxide
(poly(silicic acids) and poly(silicates)) from organic silicon
compounds (alkoxy silanes) (Cha J N, Shimizu K, Zhou Y,
Christianssen S C, Chmelka B F, Stucky G D, Morse D E (1999)
Silicatein filaments and subunits from a marine sponge direct the
polymerization of silica and silicones in vitro. Proc Natl Acad Sci
USA 96: 361-365).
[0022] Surprisingly, the inventors--first in the marine sponge S.
domuncula as a model system--could discover an enzyme (designated:
"silicase") being able to decompose both amorphous as well as
crystalline silicon dioxide.
[0023] This enzyme that is involved in the catabolism of silicon
dioxide in sponges was identified using the technique of
"differential display" of the mRNA by using the in vitro
Primmorph-cell culture system (see below).
[0024] The silicase can perform two functions: first it has the
ability (i)--in analogy to the carbonic anhydrase--to dissolve
lime-material, and (ii)--and this was surprising--also to dissolve
silicon dioxide by forming silicic acid. Thus, the silicase--as
found first in S. domuncula--is able to engage both in the
catabolism of lime-containing material as well as in the catabolism
of the silicic acid-containing spicules.
[0025] The present invention is furthermore novel in that the
silicase-gene can be induced by an increase of the
silicon-concentrations in the medium (to commonly 60 .mu.M) (see
FIG. 7).
[0026] According to a further aspect of the present invention,
generally a method for the in vitro or in vivo degradation of
amorphous or crystalline silicone dioxide (condensation products of
the silicic acid, silicates), silicones and other silicon (IV)- or
metal (IV)-compounds as well as of mixed polymers of these
compounds is provided, wherein a polypeptide or a metal complex of
a polypeptide is used for the degradation, characterized in that
the polypeptide comprises an animal, bacterial, plant or fungal
carbonic anhydrase domain that exhibits a sequence similarity of at
least 25% (see FIG. 3) to the sequence shown in SEQ ID No. 1. Until
now, it was not known that such carbonic
anhydrase-domains-containing enzymes are able to decompose such
silicates or silicones. Due to the reversibility of the process a
further aspect of the present invention relates to a method for the
synthesis of amorphous silicone dioxide (condensation products of
the silicic acid, silicates), silicones and other silicon (IV)- or
metal (IV)-compounds as well as of mixed polymers of these
compounds, wherein a polypeptide or a metal complex of a
polypeptide is used for the synthesis, characterized in that the
polypeptide comprises an animal, bacterial, plant or fungal
carbonic anhydrase domain that exhibits a sequence similarity of at
least 25% to the sequence shown in SEQ ID No. 1.
[0027] Preferred is a method according to the present invention
that is characterized in that compounds such as silicic acids,
monoalkoxy silantrioles, dialkoxy silandioles, trialkoxy silanoles,
tetraalkoxy silanes, alkyl- or aryl-silantrioles, alkyl- or
aryl-monoalkoxy silandioles, alkyl- or aryl-dialkoxy silanoles,
alkyl- or aryl-trialkoxy silanes or other metal(IV)-compounds are
used as reactants (substrates) for the synthesis. By using defined
mixtures of the compounds mixed polymers having a defined
composition can be produced.
[0028] According to a further aspect of the present invention a
formation of defined two- and three-dimensional structures can
occur by the polypeptide or a metal complex of the polypeptide or
the binding of the polypeptide or a metal complexes of the
polypeptide to other molecules or the surfaces of glass, metals,
metal oxides, plastics, biopolymers or other materials as a
template.
[0029] According to a further aspect of the present invention a
method for the modification of a silicic acid or silicon(IV)- or
metal (IV)-compound containing structure or surface is provided,
wherein a polypeptide or a metal complex of a polypeptide is used
for the modification, characterized in that the polypeptide
comprises an animal, bacterial, plant or fungal carbonic anhydrase
domain that exhibits a sequence similarity of at least 25% to the
sequence shown in SEQ ID No. 1. Preferably, the silicic
acid-containing structure or surface is present in form of a
precious stone or semi-precious stone.
[0030] Preferred is a method according to the present invention,
wherein the modification comprises a smoothing, an etching or the
production of burrows of the silicic acid or silicon(IV)- or
metal(IV)-compound-containing structure or surface by the
polypeptide or a metal complex of the polypeptide.
[0031] A further aspect of the present invention relates to a
chemical compound or silicic acid-containing structure or surface,
obtained according to a method of the present invention, in
particular in the form of a precious stone or semi-precious
stone.
[0032] A further aspect of the present invention also relates to a
polypeptide of a silicase from Suberites domuncula according to SEQ
ID Nr. 1 or a polypeptide being homologous thereto, which in the
amino acid sequence of the carbonic anhydrase domain exhibits a
sequence similarity of at least 25% to the sequence shown in SEQ ID
No. 1, a metal complex of the polypeptide, or parts thereof.
[0033] A further aspect of the present invention also relates to a
nucleic acid, in particular according to SEQ ID No. 2,
characterized in that it essentially encodes for a polypeptide
according to the invention. The nucleic acid according to the
invention can be characterized in that it is present in the form of
a DNA, cDNA, RNA or mixtures thereof and can be characterized in
that the sequence of the nucleic acid has at least one intron
and/or a polyA-sequence. Another aspect of the present invention
relates to the nucleic acid according to the invention in the form
of its complementary "antisense"-sequence.
[0034] A still further aspect of the present invention also relates
to a nucleic acid according to the invention in the form of a (a)
fusion protein-(chimeric protein) construct, (b) construct having a
separate protein-expression (protease-cleavage site) or (c)
construct having a separate protein-expression
(cassette-expression). The nucleic acid according to the invention
can be synthetically produced. Respective methods are well known in
the state of the art.
[0035] A further aspect of the present invention relates to a
vector, preferably in the form of a plasmid, shuttle vector,
phagemid, cosmid, expression vector, retroviral vector, adenoviral
vector or particle, nanoparticle or liposome, comprising a nucleic
acid according to the present invention. Furthermore, vectors for
the transfer of proteins can be provided, preferably in the form of
a nanoparticle or liposome, comprising a polypeptide according to
the present invention.
[0036] According to a further aspect of the present invention a
host cell, transfected with a vector or infected or transduced with
a particle according to the invention, is provided. Said host cell
can be characterized in that it expresses a polypeptide according
to claim 1, a metal complex of the polypeptide or parts thereof.
All know host cell-organisms are suitable as host cells, such as,
amongst others, yeasts, fungi, sponges, bacteria, CHO-cells or
insect cells.
[0037] The polypeptide according to the invention can be
characterized in that the polypeptide has been synthetically
produced or that the polypeptide or the metal complex of the
polypeptide is present in a prokaryotic or eukaryotic cell extract
or lysate. The cell extract or lysate can be obtained from a cell
ex vivo or ex vitro, for example a recombinant bacterial cell or a
marine sponge.
[0038] The polypeptide according to the invention can be purified
according to common methods known in the state of the art, and
therefore can be present essentially free of other proteins.
[0039] A further aspect of the present invention then relates to a
method for identifying of inhibitors or activators of a polypeptide
of a silicase from Suberites domuncula according to SEQ ID No. 1 or
a polypeptide being homologous thereto that in the amino acid
sequence of the carbonic anhydrase domain has at least 25% sequence
similarity to the sequence shown in SEQ ID No. 1, wherein a) a
polypeptide of a silicase from Suberites domuncula according to SEQ
ID No. 1 or a polypeptide being homologous thereto that in the
amino acid sequence of the carbonic anhydrase domain has at least
25% sequence similarity to the sequence shown in SEQ ID No. 1 is
provided, b) the polypeptide from step a) is contacted with a
potential inhibitor or activator, and c) the ability of the
polypeptide is measured to degrade or synthesize silicate or
silicones. With this method valuable substances can be identified
that are possibly suited as therapeutics (for this, see below).
Methods for the identification of such substances are known to the
person of skill, and include, for example, the use of radioactively
labeled or enzymatically labeled candidate-compounds. Methods for
measuring the activity of the silicase are described in the
following and can readily be modified by the person of skill in
view of a testing format. Thereby, an inhibitor lowers the activity
of the enzyme essentially completely, an activator induces an
activity or amplifies it above the baseline.
[0040] According to an alternative of the method the polypeptide of
a silicase from Suberites domuncula according to SEQ ID No. 1 or a
polypeptide being homologous thereto that in the amino acid
sequence of the carbonic anhydrase domain has at least 25% sequence
similarity to the sequence shown in SEQ ID No. 1 can be provided in
vivo, in a cellular extract or lysate or in purified form.
[0041] A still further aspect of the present invention relates to a
method for producing a pharmaceutical composition, comprising a)
identifying of an inhibitor or activator according to claim 25 or
26 and b) mixing of the identified inhibitor or activator with a
pharmaceutically acceptable carrier or excipient. By means of this
composition, valuable pharmaceutics are provided that, such as for
example the polypeptide or a nucleic acid or pharmaceutical
composition can be used for the prevention or therapy of silicosis.
Preferred is a use, wherein the prevention and therapy of silicosis
occurs by dissolving of quartz crystals. Furthermore, the use of
polypeptide or a nucleic acid or pharmaceutical composition
according to the invention for the resorption or for modulating the
resorbability of silicones and silicone implants can take place.
Finally, the present invention can be used for transfecting cells
with nucleic acids according to the invention for the resorption or
for modulating the resorbability of silicones and silicone
implants. The above indicated uses and the methods therefore are
known to person of skill and can readily be adjusted to the needs
and requirements as present here.
1.5. Cloning of the Gene Encoding the Silicase
[0042] By use of the technique of the "Differential Display" a cDNA
was identified encoding for a carbonic anhydrase. For carbonic
anhydrases until now only an involvement in the regulation of the
pH, the HCO.sub.3.sup.--reabsorption and the CO.sub.2-expiration
was known, but not an involvement in the--yet unknown--enzymatic
dissolution of silicon dioxide-materials.
[0043] The cDNA encoding for the silicase from the marine sponge S.
domuncula (designated: SDSIA) as well as the polypeptide derived
from the nucleotide sequence (designated: SIA_SUBDO) have the
following properties. Length of the cDNA: 1395 nucleotides (nt);
open reading frame: from nt.sub.122-nt.sub.124 to
nt.sub.1259-nt.sub.1261 (Stop codon); length of the polypeptide:
379 amino acids; relative molecular mass (M.sub.r) of the
polypeptide: 43131; isoelectric point (pI): 6.5 (calculated with:
PC/GENE (1995) Data Banks CD-ROM; Release 14.0. IntelliGenetics,
Inc. Mountain View, Calif.).
[0044] The Northern-blot-analysis with the sponge SDSIA-clone as a
probe results in a band of 1.5 kb.
[0045] FIG. 2 (below) shows the nucleotide sequence of the
sponge-silicase-cDNA--identified with the aid of the differential
display technique-, and FIG. 2 (above and below) as well as FIG. 3A
show the polypeptide derived from the nucleotide sequence of the
sponge-silicase (SIA_SUBDO).
[0046] The derived amino acid sequence of the sponge-silicase has a
large similarity to the amino acid sequences of the carbonic
anhydrase-family. Until now, no more than seven isoenzymes of
carbonic anhydrases were identified in humans (Sun M K, Alkon D L
(2002) Carbonic anhydrase gating of attention: memory therapy and
enhancement. Trends Pharmac Sci 23: 83-89). The "Expect value" [E]
(Coligan J E, Dunn B M, Ploegh H L, Speicher D W, Wingfield P T
(2000) Current protocols in protein science. John Wiley & Sons,
Chichester) of the sponge-silicase with the human carbonic
anhydrase II (CAH2_HUMAN; P00918) is 2e.sup.-29. The
eukaryotic-type-carbonic anhydrase domain (PFAM00194
[www.ncbi.nlm.nig.gov]) in the sponge-silicase is found in the
amino acid-region of aa87 to aa335 (FIG. 3A). The alignment of the
sponge-silicase with the human carbonic anhydrase II shows that
most of the characteristic amino acids that form the
eukaryotic-type-carbonic anhydrase-signature (Fujikawa-Adachi K,
NishimoriI, Taguchi T, Yuri K, Onishi S (1999) cDNA sequence, mRNA
expression, and chromosomal localization of human carbonic
anhydrase-related protein, CA-RPXI. Biochim Biophys Acta 1431:
518-524; Okamoto N, Fujikawa-Adachi K, Nishimori I, Taniuchi K,
Onishi S (2001) cDNA sequence of human carbonic anhydrase-related
protein CA-RP X and XI in human brain. Biochim Biophys Acta 1518:
311-316) are also present in the sponge-silicase. Nevertheless, in
the sponge-sequence the amino acid residues 192 (alanine), 205
(phenylalanine) and 207 (phenylalanine) are replaced (FIG. 3A).
[0047] The carbonic anhydrases constitute a family of zinc
metal-enzymes that are involved in the reversible hydration of
CO.sub.2 (Sly W S, Hu P Y (1995) Human carbonic anhydrases and
carbonic anhydrase deficiencies. Annu. Rev. Biochem. 64: 375-401).
The three conserved histidine residues are found in the silicase at
the amino acids aa181, aa183, and aa206 (FIG. 3A).
1.6. Phylogenetic Analysis of the Silicase
[0048] FIG. 3B shows the position of the sponge-silicase amongst
different elected representatives of the carbonic anhydrase-family
(phylogenetic tree; "rooted tree" with the bacterial carbonic
anhydrase-sequences of Neisseria gonorrhoeae). The sponge-silicase
together with the carbonic anhydrase of Caenorhabditis elegans form
the base for the carbonic anhydrases of the other metazoes. The
metazoic enzymes are separated from the plant-enzymes and also from
the bacterial enzymes.
2. Production of Silicase
[0049] Die silicase can be purified from tissues or cells or can be
recombinantly produced.
2.1. Purification of the Silicase from Natural Sources
[0050] All steps are performed at 4.degree. C. For the purification
of the silicase (or carbonic anhydrase or carbonic
anhydrase-related enzyme) the homogenized tissue is--for example in
a Tris-SO.sub.4/Sodiumsulfate-buffer (pH 8.7)--(or are cells that
are homogenized in this buffer) centrifuged and an affinity
chromatography-matrix such as for example CM-Bio-Gel A, coupled
with p-aminomethyl benzene sulfonic acid amide is added to the
supernatant as obtained. Then, the suspension is incubated on a
rotating shaker (for example for 24 h). The affinity gel is then
collected by suction via a glass filter, and washed with a buffer
(for example 0.1 M Tris-SO.sub.4, pH 8.7 containing 0.2 M
Na.sub.2SO.sub.4, 1 mM benzamidine and 20% glycerol). Subsequently
it is suitable to add a second wash step with the same buffer at a
lower pH (for example pH 7.0) in order to remove unspecifically
bound proteins. The gel is then transferred into a column and
washed with the same buffer (pH 7.0). For an elution of the enzyme
for example a 0.1 M Tris-SO.sub.4-buffer, pH 7,0, containing 0.4 M
NaN.sub.3, 1 mM benzamidine and 20% glycerol can be used. The
eluted enzyme protein is then dialysed against for example a 10 mM
Tris-SO.sub.4-buffer, pH 7.5, containing 1 mM benzamidine, and
thereafter added on an ion exchange column (for example
DEAE-Sephacel) which, for example, has been equilibrated with 10 mM
Tris-SO.sub.4-buffer, pH 7.5. After the washing with the same
buffer the enzyme is eluted by applying a linear salt-gradient (for
example 0 to 0.1 M Na.sub.2SO.sub.4) and collected. With the aid of
this procedure the silicase, amongst others, can be purifies from
the sponge S. domuncula.
2.2. Production of the Recombinant Silicase
2.2.1. Cloning of the cDNAs from Marine Sponges
[0051] Performing the technique of the "Differential Display" of
the mRNA/transcripts is state of the art (Muller W E G, Krasko A,
Skorokhod A, Bunz C, Grebenjuk V A, Steffen R, Batel R, Muller I M,
Schroder H C (2002) Histocompatibility reaction in the sponge
Suberites domuncula on tissue and cellular level: central role of
the allograft inflammatory factor 1. Immunogenetics 54,48-58).
Total-RNA is isolated from control cultures (held at a low
silicon-concentration of 5 pM) as well as from cultures treated in
the presence of 60 .mu.M silicon by using the TRIzol reagent
(GibcoBRL). The synthesis of the first cDNA-strand is performed
with "anchored" oligo (dT)-primers and AMV reverse transcriptase
according to the protocol of the manufacturer (Promega). After the
synthesis of the first strand the resulting cDNA is diluted tenfold
with H.sub.2O, and a aliquot part thereof (2 .mu.l) is subjected to
the polymerase-chain-reaction (PCR). The reaction is performed in a
volume of 20 pl after the addition of the "arbitrary" primers 1
(5'-GTGATCGCAG-3') or 2 (5'-CTTGATTGCC-3') as well as of 2 .mu.M
dNTP, T.sub.11GC, 5 units BioThem Polymerase (Genecraft), and
[.alpha.-32P] dATP performed. The following reaction conditions
have could be found as suitable for the PCR: initial denaturation
at 95.degree. C. for 5 minutes, then 40 amplification cycles each
at 95.degree. C. for 20 seconds, 42.degree. C. for 120 seconds,
72.degree. C. for 30 seconds, followed by a final incubation of 10
minutes at 72.degree. C. The samples are then separated in a 5%
polyacrylamide gel (in 1.times.TBE). After the run the gel is dried
and exposed for 4 days to an x-ray film. The interesting bands that
are identified in the autoradiogram are cut out, boiled for 15
minutes in 200 ul H.sub.2O, chilled on ice and centrifuged for 10
minutes at 14000.times.g. The resulting supernatants are
supplemented with the same volume of 10 M ammonium acetate, 20
.mu.g/ml tRNA and precipitated with 2.5 volumes of ethanol at
-80.degree. C. over night. The cDNA-pellets are washed three times
in 75% ethanol, and dissolved in 20 .mu.l H.sub.2O.
[0052] Approximately 2 .mu.l of the eluted bands are re-amplified
in 50 .mu.l-reaction-preparations by using the above described
primers under the same conditions, are subcloned in a pGEM-T-vector
(Promega), and sequenced.
[0053] Those transcripts are selected that are differentially
expressed, i.e. that are additionally contained in the gels with
the RNAs of cells that have been treated with 60 .mu.M silica (FIG.
1). The identified cDNAs/transcripts are compared with sequences
contained in the BLAST data base. In the example given in FIG. 1
the following molecules showed the largest relation:
Calcium/Calmodulin-dependent protein kinase (CaM Kinase)II gamma
(XM.sub.--044349; Expect value [E]:1e.sup.-16); hypothetic protein
(XP.sub.--101359,E 1,6); MUC3B mucin (AJ291390, E 0,20); hypothetic
protein (XP.sub.--067115, E 5,9); hypothetic protein
(XP.sub.--090138, E 2,9); ATP-binding cassette, subfamily A member
4 (XM.sub.--001290, E 1,6); polypeptide similar to the zinc finger
protein 91 (XM.sub.--091947, E 3,1); hypothetic protein
(XP.sub.--104250, E 0,48), hypothetic protein (XP.sub.--169372, E
8,6); hypothetic protein (XP.sub.--104250, E 4,1), hypothetic
protein (XP.sub.--098020, E 3,3) and hypothetic protein
(XP.sub.--169372, E 8,6).
[0054] In addition to these sequences the silicase was identified
as additional transcript and analyzed in more detail.
[0055] The silicase gene can also be identified from
cDNA-libraries, e.g. in ZapExpress and in Escherichia coli XL1-Blue
MRF', with suitable degenerated primers by means of the
PCR-technique; for this, the corresponding vector-specific primers
are used. The synthesis products as obtained are used for screening
in the den respective cDNA-libraries. Then, the identified clones
are subcloned in a vector (for example pGem-7) and subsequently
sequenced.
2.2.2. Expression and Isolation of the Recombinant Silicase
[0056] The production of the recombinant silicase (designated:
rSIA_SUBDO) preferably occurs in E. coli. Nevertheless, also the
production in yeasts and mammalian cells is possible and was
successfully done. In the following as an example the expression of
the SDSIA-gene of S. domuncula in E. coli using the "GST
(glutathione-S-transferase) fusion"-system (Amersham) described. In
the example two inserts are used in order to eliminate potential
effects of signal peptides during the expression; one insert
comprises the whole derived protein (long form; from amino acid
aa.sub.1 to the amino acid aa.sub.379) and the other insert only
the amino acids aa.sub.96 to aa.sub.379 (short Form) (FIG. 3A). The
corresponding clones are designated as SDSIA-I and SDSIA-s. They
are cloned in a corresponding vector, e.g. into the plasmid
pGEX-4T-2, containing the glutathione-S-transferase (GST)-gene of
Schistosoma japonicum. Also other expression vectors have proven
suitable. After transformation von E. coli the expression of the
silicase is usually induced by IPTG
(isopropyl-.beta.-D-thiogalactopyranoside), and performed in the
presence of 1 mM ZnSO.sub.4 for 4 or 6 hours at 37.degree. C.
(Ausubel F M, Brent R, Kingston R E, Moore D D, Smith J A, Seidmann
J G, Struhl K (1995) Current Protocols in Molecular Biology. John
Wiley and Sons, New York). The obtained GST-fusions proteins with
the designation SIA_SUBDO-I (long form; M.sub.r 69 kDa) or rSIA
SUBDO-s (short form; M.sub.r 58 kDa) are, e.g. purified by affinity
chromatography on glutathione-Sepharose 4B. For a separation of the
glutathione-S-transferase from the recombinant sponge-silicase the
fusions proteins are cleaved with thrombin (10 units/mg). The
proteins are then subjected to gel electrophoresis in the presence
of 2-mercaptoethanol. The gel electrophoresis can be performed in
10% polyacrylamide gels with 0.1% NaDodSO.sub.4 (PAGE). The gels
are stained with Coomassie Brillant blue.
[0057] After the cleavage, purification, and subsequent PAGE the
long form (rSIA_SUBDO-I [43 kDa]) and the short form (rSIA_SUBDO-s
[32 kDa]) of the recombinant proteins are obtained (FIG. 4).
2.2.3. Expression and Isolation of the Recombinant Silicase from
Other Organisms
[0058] In agreement with the above described strategy, the
isolation, cloning, and expression of the silicase-cDNA from other
organisms can also be performed, for example from (silicon
dioxide-producing) diatoms (e.g. Cylindrotheca fusiformis). The
method of obtaining diatoms in axenic cultures is state of the art
(Kroger N, Bergsdorf C, Sumper M (1996) Europ J Biochem 239:
259-264).
2.3. Isolation and Purification of the Silicase by Means of
Antibody
[0059] Following extraction or partial purification according to
the above described methods the silicase is purified on an
antibody-affinity matrix. The affinity matrix is produced in that a
silicase-specific antibody is immobilized on a solid phase
(CNBr-activated sepharose or other suitable carrier). As antibody,
monoclonal or polyclonal antibodies against the silicase are used
that are produced according to standard methods (Osterman L A
(1984) Methods of Protein and Nucleic Acid Research, Vol 2,
Springer-Verlag, Berlin). The coupling of the antibody to the
matrix of the column is done in accordance with the instructions of
the manufacturer (Pharmacia). The elution of the pure silicase
occurs by a change of pH or ionic strength.
3. Determination of the Silicase-Activity
[0060] In the following only the activities are given that have
been found for the short form of the recombinant sponge-silicase
(rSIA_SUBDO-s).
3.1. Carbonic Anhydrase-Activity
[0061] For determining the carbonic anhydrase-activity of the
rSIA_SUBDO-s, an assay can be used that is based on the hydrolysis
of p-nitrophenylacetate (Armstrong J M, Myers D V, Verpoorte J A,
Edsall J T (1966) Purification and properties of human erythrocyte
carbonic anhydrase. J Biol Chem 241: 5137-5149). 0.5 ml of a 3 mM
p-nitrophenylacetate-solution (Sigma) are mixed with 0.05 ml of a
0.3 mM Tris-HCl-buffer (pH 7.6). After pre-incubation at 25.degree.
C. for 5 minutes 50 .mu.l of the recombinant silicase (rSIA_SUBDO)
are added and the increase of the extinction at 348 nm is
determined over a period of 5 minutes.
[0062] FIG. 5 shows that the activity of the recombinant silicase
depends from the concentration of the enzyme in the assay. The
activity of the enzyme is given optical density (OD)-units per
minute. The addition of 1 .mu.g silicase per assay (0.56 .mu.l)
resulted in an activity of 0.005 OD.sub.348 nm, that increased with
increasing protein concentration up to 0.04 OD.sub.348 nm.
3.2. Silicase-Activity
[0063] As substrate (amorphous silicon dioxide) for the silicase,
for example, spicules of S. domuncula are suitable. The spicules
can be obtained from sponge tissue by 12-hour incubation in the
presence of ethylene diamine tetraacetic acid (20 mM, in PBS;
PBS=phosphate buffer-salt-solution, consisting of 1.15 mM
KH.sub.2PO.sub.4, 8.1 mM Na.sub.2HPO.sub.4, 137 mM NaCl and 2.7 mM
KCl). After washing with distilled water and with ethanol (two
times) the spicules are dried (56.degree. C.) and then grinded to a
powder in a mortar.
[0064] The silicase-activity can be determined as follows.
Commonly, 100 .mu.g of the dried spicules (powder) are added to a
suitable buffer, such as 50 mM Tris-HCl-buffer (pH 7.2; 10 mM
DL-dithiothreitol, 100 mM NaCl) and 0.5 mM ZnSO.sub.4 in 2 ml
Eppendorf-tubes. Then, usually 50 .mu.l of the recombinant silicase
are added, and incubated at 25.degree. C. (the incubation is
possible also at other temperatures between 5.degree. C. and about
65.degree. C.). The average incubation time is 60 minutes. For a
quantitative determination of the amount of dissolved silicon
dioxide, the non dissolved spicules are spun off (14000.times.g; 15
minutes; 4.degree. C.). The released soluble silicic acid can be
quantitatively determined e.g. with the aid of a
molybdenum-supported determination methods such as e.g. the
colorimetric "Silicon Test" (Merck; 1.14794). In this case, the
amount of silicic acid is calculated from the values of extinction
at 810 nm based on a calibration curve with a silicon standard
(Merck 1.09947).
[0065] FIG. 5 shows that the recombinant silicase catalyses the
degradation (dissolution) of amorphous silicon dioxide. Commonly,
at an enzyme concentration of 1 .mu.g recombinant silicase, 3 ng
silicic acid/assay per assay are released. At higher protein
concentrations (3 or 10 .mu.g/assay) the release of silicic acid is
20 or 43 ng/assay.
3.3. Silicase-Activity in Escherichia coli-Lysate
[0066] The silicase-activity can also be determined in lysates of
E. coli that were transformed with the SDSIA-gene of S. domuncula
(in the following example, the short form was used; =SDSIA-s) using
the "GST fusion"-system. In the experiment as shown in table 1
sponge-spicules (needles; 1 mg) were incubated at different
temperatures with 1.5 ml lysate, to which 1 mM ZnCl.sub.2 and 0.1 M
NaCl were added. After 1, 3, 6, and 24 h the samples were
denaturated by heating to 95.degree. C. for 10 min (for
inactivating the silicase), and the incubated with proteinase K (30
Units/ml) at 37.degree. C. for 1 h. A subsequently centrifugation
(5 min, 14000 rpm) followed, and the molybdenum-assay (kit of the
company Merck; see above) was used for a determination of the
released silicate. It was found that at 4.degree. C. only a very
small amount of silicate was released, nevertheless at room
temperature (22.degree. C.) and 56.degree. C. up to 3.4 and 4.1
ng/ml (24 h), respectively.
[0067] A slightly lower amount of released silicate could also be
determined in lysates from non-transformed E. coli indicating the
also in bacterial cell extracts a marked silicate-decomposing
activity is present. TABLE-US-00001 TABLE 1 Silicase-activity in
lysates of transformed (+) and non-transformed (-) E. coli at
different temperatures of incubation. For the transformation the
short form of SDSIA-gene of S. domuncula (=SDSIA-s) was used. The
release of silicate was determined after a time of incubation of 1,
3, 6, and 24 hours. Released silicate (ng/ml) Temperature 1 h 3 h 6
h 24 h 4.degree. C. (-) 0.113 0.124 0.242 0.303 4.degree. C. (+)
0.110 0.140 0.526 0.828 22.degree. C. (-) 0.197 0.415 1.467 2.068
22.degree. C. (+) 0.528 0.540 1.939 3.409 56.degree. C. (-) 0.345
1.009 1.824 2.447 56.degree. C. (+) 1.542 1.747 2.275 4.092
3.4. Silicase-Activity of Commercial Carbonic Anhydrases
[0068] A silicase-activity can not only be measured in the
sponge-enzyme, but surprisingly also in commercial carbonic
anhydrases. Table 2 shows the release of silicate from skeletons of
diatoms (silicate-scaffolds of diatoms) as well as from sand by a
commercial carbonic anhydrase-preparation (from bovine
erythrocytes; company Calbiochem). In the experiment, the
silicate-samples were first washed twice with water and twice with
ethanol and then dried. Subsequently, the samples were suspended in
50 mM Tris-HCl-buffer, pH 7.6 (1 mg/ml) and dispersed in 2
ml-Eppendorf-tubes (100 .mu.l per reaction tube; =100 pg silicate
per reaction tube). Then, 1.4 ml bovine carbonic anhydrase (10
units; company Calbiochem) in 50 mM Tris-HCl-buffer, pH 7.6 (with
and without 1 mM ZnCl.sub.2) were added per reaction tube. The
tubes were incubated at room temperature (22.degree. C.) by shaking
for 24 h. Then, the preparations were centrifuged (14000.times.g,
15 min, 4.degree. C.). The silicate-content in the supernatant was
determined with the aid of the molybdenum-assay of the company
Merck (see above). TABLE-US-00002 TABLE 2 silicase-activity of a
commercial carbonic anhydrase-preparation (company Calbiochem) with
and without addition of ZnCl.sub.2. The release of silicate was
determined after an incubation time of 24 hours. Released silicate
(ng/ml) Diatoms Sand Minus ZnCl.sub.2 0.0018 0.0036 Plus ZnCl.sub.2
2.0233 0.0359
3.5. Reversibility of the Silicase-Activity
[0069] The silicase-reaction in principle is reversible. Thus, the
reaction can also be used for the synthesis of amorphous silicon
dioxide or silicones. For the silicase-mediated synthesis also
alkyl or aryl substituted alkoxy compounds of silicon(IV), such as
tetraalkoxysilanes, trialkoxysilanoles, dialkoxysilandioles,
monoalkoxysilantrioles, alkyl or aryl trialkoxysilanes, alkyl or
aryl dialkoxysilanoles or alkyl or aryl monoalkoxysilandioles can
be used. In addition, mixtures of these substrates are polymerised.
Therefore, also mixed polymers can be produced.
4. Ligation of the cDNA for Silicase with One or Several cDNA(s)
for Other Proteins
4.1. Production of Silicase-Fusion Proteins
[0070] For a production of fusion proteins with the silicase a
suitable expression vector (for example pQE-30-vector; Qiagen) is
used. The silicase-cDNA--having e.g. a Ban HI-restriction site at
its 5'-terminus and e.g. a Sal I restriction site at its
3'-terminus--is produced. The stop-codon in the silicase-cDNA is
removed. For this, the PCR-technique is used, and for the
amplification primers, which have the respective restriction sites,
are used. The cDNA for the second protein is obtained accordingly,
whereby at the 5'-terminus the same cutting site is present as at
the 3'-terminus of the silicase-cDNA (Sal I in the example) and one
that is different from the other is present at the 3'-terminus
(e.g. a Hind III-site). If internal restriction sites are present
in the respective cDNAs, alternative restriction enzymes can be
used. In addition, linkers between both cDNAs can be used.
[0071] Both cDNAs are ligated according to the common method,
purified and ligated into the pQE-30-vector. The ligation takes
place following the histidine-tag (about 6 histidine-codons). The
expression and purification of the fusion protein using, e.g. the
histidine-tag being present at the recombinant protein, can be
performed on respective affinity columns, e.g. a Ni--NTA-matrix
(Skorokhod A, Schacke H, Diehl-Seifert B, Steffen R, Hofmeister A,
Muller W E G (1997) Cell Mol Biol 43: 509-519).
4.2. Separate Expression I (Protease-Cleavage Site)
[0072] As an alternative to the method at 4.1. a protease-cleavage
site (such as, for example, an enterokinase-site) can be cloned
between the cDNA for the silicase and the cDNA for an additional
protein. In this case a codon for a novel start-methionine can be
inserted in front of the encoding region of the gene for the
additional protein. Following expression and purification the
fusion protein is proteolytically cleaved. Now, both proteins are
present separately.
4.3. Separate Expression II (Cassette-Expression)
[0073] As an alternative, both proteins can be expressed separately
on one construct. For this, in an expression-vector the
silicase-gene is following the his-tag. At the end of the
silicase-cDNA, a stop-codon is inserted. A ribosome-binding site
with a codon for a start-methionine is cloned between the cDNA for
the silicase and the cDNA for the additional protein. Again, a
his-tag is positioned in front of the cDNA for the additional
protein. Also this gene is provided with a stop-codon.
[0074] The his-tags can be deleted, if the proteins are used fort
he functional analysis in the respective host cells.
4.4. Extensions
[0075] For the expression described at 4.1 to 4.3 bacterial as well
as eukaryotic cells can be used.
[0076] The expression described at 4.1 to 4.3 can also be used for
three and more open reading frames.
5. The Model System for the Synthesis/the Degradation of Biogenic
Silicon Dioxide: Primmorphs
5.1. Primmorphs
[0077] A patent application was filed for the Primmorph-system (DE
19824384. Herstellung von Primmorphe aus dissoziierten Zellen von
Schwammen, Korallen und weiteren Invertebraten: Verfahren zur
Kultivierung von Zellen von Schwammen und weiteren Invertebraten
zur Produktion and Detektion von bioaktiven Substanzen, zur
Detektion von Umweltgiften und zur Kultivierung dieser Tiere in
Aquarien und im Freiland. Inventors and applicants: Muller W E G,
Brummer F).
[0078] Primmorphs are aggregates that consist out of proliferating
and differentiating cells (Muller W E G, Wiens M, Batel R, Steffen
R, Borojevic R, Custodio M R (1999) Establishment of a primary cell
culture from a sponge: Primmorphs from Suberites domuncula. Marine
Ecol Progr Ser 178: 205-219). Primmorphs are formed from sponge
single cells that are obtained from sponge tissue after
dissociation in Ca.sup.2+ and Mg.sup.2+ free, EDTA containing
artificial seawater.
[0079] Aggregates are formed from the sponge single cells after
transfer into Ca.sup.2+ and Mg.sup.2+-containing seawater that
after 3 days reach a size of 1 mm, and after 5 days Primmorphs with
a diameter of about 5 mm.
[0080] The Primmorphs are surrounded by epithelium-like cells, the
pinacocytes. The cells within the Primmorphs are primarily
spherical cells, in addition, amoebocytes and archaeocytes are
present.
5.2. Effect of Silicon on the Formation of Spicules
[0081] The Primmorph-system of sponges, e.g. S. domuncula, can be
used fort he examination of the formation or dissolution of
spicules.
[0082] For this, Primmorphs are cultivated for 8 days in seawater
that was supplemented with 30 uM Fe(+++) (added as citrate) and 10%
RPM11640-medium. The silicon-concentration in seawater/medium is 5
.mu.M. After 8 days the Primmorphs are either further incubated in
this medium or transferred in a medium containing 60 .mu.M silicon
(the silicon-concentration being optimal for the formation of
spicules; added as Na-hexafluorosilicate), and cultivated for 1 or
3 days.
[0083] Primmorphs that were cultivated without the addition of
silicon primarily show a round, spherical shape.
[0084] FIG. 6A shows that most of the Primmorphs after additional
3-day culture in the presence of 60 .mu.M silicon become ovally
shaped. In the presence of silicon, the Primmorphs start with the
formation of spicules. Partially, the synthesis of long (>100
um) spicules can be observed (FIG. 6B), nevertheless, more often
smaller spicules (30 um) are found (FIG. 6D). In the absence of
silicon, no spicules are present (FIG. 6C).
5.3. Silicon-Responsive Genes
[0085] In Primmorphs of S. domuncula the expression of the
silicase-gene is up-regulated in the presence of silicon. In
parallel also the expression of the following genes is increased:
silicatein, collagen, myotrophin and isocitrate-dehydrogenase.
[0086] The expression of the silicase-gene can be determined by
Northern-blotting using methods that are state of the art were, for
example, used for the determination of the expression of silicatein
and collagen (Krasko A, Batel R, Schroder H C, Muller I M, Muller W
E G (2000) Expression of silicatein and collagen genes in the
marine sponge Suberites domuncula is controlled by silicate and
myotrophin. Europ J Biochem 267: 4878-4887).
[0087] In the experiment shown in FIG. 7 the Primmorphs either
maintained untreated or were incubated with 60 .mu.M silicon for 1
to 3 days. Then, the RNA was extracted. An amount of each 5 .mu.g
total-RNA was electrophoretically separated on a 1%
fornaldehyde/agarose-gel, blottet onto a Hybond-N+ Nylon-membrane
in accordance with the instructions of the manufacturer (Amersham).
The hybridisation was done with 400 to 600 bp sized segments of the
following probes: SDSIA (encodes for silicase), SDSILICA (encodes
silicatein), and SDIDH (encodes for the .alpha.-subunit of the
isocitrate-dehydrogenase). The probes were labelled with the
PCR-DIG-probe-synthesis kit in accordance with the instructions of
the manufacturer (Roche). After washing the DIG-labelled nucleic
acid was detected with anti-DIG Fab fragments (conjugated with
alkaline phosphatase; dilution: 1:10000), and visualized with the
aid of the chemoluminescence technique using CDP (Roche), the
chemoluminescence-substrate of the alkaline phosphatase.
[0088] FIG. 7 shows the Northern-blots that were obtained. It can
be seen that the genes for the silicase and silicatein are strongly
up-regulated in response to higher silicon concentrations.
Furthermore also the gene for isocitrate-dehydrogenase (encodes for
an enzyme being involved in the citric acid cycle) are up-regulated
indicating that the formation of amorphous silicon dioxide requires
an increased metabolic rate of the cells.
6. Mode of Action of the Silicase
[0089] The finding obtained by the sequence comparisons, that the
silicase is a member of the family of carbonic anhydrases
(carbonate-hydrolase; EC 4.2. 1.1), was surprising.
[0090] These enzymes catalyse the reversible hydration of carbon
dioxide (FIG. 8 [1]). Carbon dioxide is converted into
HCO.sub.3.sup.- and H.sup.+ by the carbonic anhydrase.
[0091] The silicase indeed also exhibits a carbonic
anhydrase-activity, as could be shown with a colorimetric assay
(Armstrong J M, Myers D V, Verpoorte J A, Edsall J T (1966)
Purification and properties of human erythrocyte carbonic
anhydrase. J Biol Chem 241: 5137-5149). Accordingly, it is possible
that the silicase causes a change of the pH because of the
conversion of CO.sub.2 into HCO.sub.3.sup.- (FIG. 8 [1]). This
allows for an etching of lime substrates, but not of silicon
dioxide-materials, whose solubility increases with increasing but
not lowering pH.
[0092] It is known that some species of sponges such as species of
the genus Cliona are able to dissolve calcium carbonate and to
burrow holes into calcite/aragonite-substrates (Rutzler K, Rieger G
(1973) Sponge burrowing: fine structure of Cliona lampa penetrating
calcareous substrata. Mar Biol 21: 144-162).
[0093] Nevertheless the silicase-activity of the enzyme was unknown
and surprising.
[0094] It is known that three histidine residues are involved in
the mode of action (carbonic anhydrase-activity) of the carbonic
anhydrase that bind to a divalent zinc ion; accordingly, the
following mode of action can be formulated for the
silicase-activity (FIG. 8 [2]). In the silicase of S. domuncula the
histidine residues are found in the derived polypeptide at the
amino acid positions aa.sub.181, aa.sub.183 and aa.sub.206 (FIG.
3A). In water (Lewis-base) a hydroxide anion is formed that is
bound to the Zn.sup.2+ (Lewis-acid). This performs a nucleophilic
attack at one of the silicon atoms that are linked one with the
other by oxygen atoms (FIG. 8). In the next step the zinc-complex
binds to the silicon atom by cleaving of the oxygen bond. Under
consumption of H.sub.2O finally a free silicic acid is released the
initial zinc(II)-bound hydroxide anion is formed again.
7. Uses of the Silicase and Silicase-Fusion Proteins
[0095] For the recombinant silicase, the silicase as purified from
different sources, and the silicase-fusion proteins a series of
different industrial and technical uses are found, namely: 1.) Use
for the surface modification of biomaterials (improvement of the
biocompatibility). Surface-modified biomaterials find use amongst
others for influencing of cellular adhesion and growth, for
modifying the blood compatibility or for controlling the
protein-adsorption (e.g. reduction of the adsorption of contact
lenses). A literature compendium can be found in: Ratner B D et al
(eds.) Biomaterials Science--An Introduction to Materials in
Medicine. Academic Press, San Diego, 1996. One problem consists in
the fact that the conditions used for the production of these
modifications often have a deleterious (destructive) effect on the
used biomaterials. A "mild" and biomaterial protective method
compared to the physical/chemical methods as used is represented by
a modification of the surfaces that is solely based on
biochemical/enzymatic reactions that becomes possible with the aid
of the method according to the invention (silicase-mediated
enzymatic degradation and--as reversible reaction--enzymatic
synthesis of SiO.sub.2 or siloxane containing of surfaces with the
aid of the recombinant/purified silicase). In particular, also a
use of the recombinant or silicase as purified from natural sources
in the production of surface modifications (during coating) of
silicone-materials, such as silicone breast implants,
endo-prostheses or metal-implants (improvement of the connection
between bones and metal-implants, biologization of the
metal-implants) as well as contact/plastic lenses is resulting.
Further uses relate to the coating of collagen, that is used as
bone replacement material, and of collagen-fleece that are, e.g.,
used for the "tissue engineering".
[0096] Here, the goal is the increase of stability and the porosity
as well as die improvement of the resorbability.
[0097] 2.) Use for the production of novel biomaterials such as
bone replacement materials or dental replacement materials by a
co-synthesis of poly(silicates), silicones and mixed polymers.
[0098] 3.) Use for the surface modification (treatment of
contact-zones) of (silicon)-semi conductors or silicon-chips.
[0099] 4.) Use for the modification or for the synthesis of
nanostructures from amorphous silicon dioxide. By means of the
recombinant silicase, the recombinant silicase-fusion proteins or
the purified silicase it becomes possible to modify or to
synthesize specific two- and three dimensional structures from
amorphous silicon dioxide in the nanoscale. The structures as
formed can be employed in the nanotechnology.
[0100] 5.) Use for the surface modification of silicon-containing
precious stones and semi-precious stones. Agate, jasper, and onyx,
amongst others, belong to the amorphous or fine crystalline
modifications of the SiO.sub.2. Due to the possibility to modify
the surface of these minerals with the aid of the silicase under
controlled conditions, the use of the methods according to the
invention in the production or processing of these precious
stones/semi-precious stones is resulting. Here, also the
possibility to selectively introduce foreign molecules/atoms is
resulting.
[0101] 6. Use in the modification or synthesis von silicon-organic
compounds including sila-pharmaceutics. For an overview about the
production of silicon-organic compounds as a basis for so called
sila-pharmaceutics (pharmaceutics wherein C is replaced by Si),
see: Chem. unserer Zeit 14, 197-207 (1980), as well as: Bioactive
Organo-Silicon Compounds (Topics Curr. Chem. 84), Berlin, Springer
1979). By means of the method according to the invention novel,
enzymatic pathways for the modification or synthesis of such
compounds are possible.
8. Uses of the Silicase and Silicase-Fusion Proteins for the
Prevention and Therapy of Silicosis (Quartz Dust-Lung Disease)
8.1. Silicosis
[0102] Crystalline silicic acid (silicon dioxide), in the form von
quartz, tridymite or cristobalite, is most likely one of the most
important hazardous compound during work. The severity of the
adverse effect on the health and the diversity of the possible
sources of exposure are known for a long time. Due to the
widespread occurrence of crystalline silicon dioxide in the crust
of the earth and the common use of materials containing it, in
particular workers in a variety of different industrial businesses
are exposed to crystalline silicon dioxide. It can be assumed that
in agriculture, in mining, in the glass and glass-fiber industry,
as well as in the production of cement, in the production of
ceramics, in casting houses, in the production of colors, soaps and
cosmetics or in the dental manufacture/repair millions of employees
are regularly exposed to crystalline silicon dioxide. According to
the "American Thoracic Society" silicon dioxide word-wide is one of
the major causes of lung disease. Thus, a large need exists fort he
development of strategies for the prevention and therapy.
[0103] It is known that inhaled crystalline silicon dioxide causes
lung fibrosis (silicosis) and lung cancer. The silicosis is a
malign pneumoconiosis that is caused by an accumulation of silicon
dioxide-particles in the tissue of the lung, is characterized by
the occurrence of silicotic nodules. A rational therapy of this
disease leading to a severe disabling does not exists.
[0104] A major reason for the toxicity of dust-like crystalline
silicon dioxide can be found in the fact that the lung is not able
to eliminate the inhaled dust particles. The silicon
dioxide-particles remaining in the lung tissue lead to inflammatory
reactions and to the formation of cell-toxic cytokines, proteases,
and reactive oxygen radicals. A continuation of these phenomena
results in a proliferation of connective tissue results with an
increased formation of collagen in the lung, leading to the
generation of pneumoconiosis.
[0105] In general, silicosis is developing very slowly over the
course of decades. It is a progressive disease that can not be
cured. It is first apparent by dyspnoea, dry cough, and sharp pain
in the chest. A congestion of the heart and an obstruction of
respiration and circulation finally lead to death. The average
period of time between the exposition to dust and the occurrence of
the silicosis is found at about 20 years. A dangerous complication
of the silicosis is the silico-tuberculosis The mechanism leading
to the development of lung cancer by crystalline silicon dioxide is
only understood to a very limited extent.
[0106] Silicosis is the most common dust-lung disease amongst the
industrial diseases.
[0107] The mean total costs for a silicosis-patient are in the
order of about 130.000 Euro.
8.2. Therapeutics/Protective Agent in Silicosis
[0108] The silicase that is involved in the dissolution of biogenic
silicon dioxide can be used as therapeutic/protective agent for the
treatment of silicosis.
[0109] The silicase is not only able to dissolve amorphous but also
crystalline silicon dioxide (quartz crystals).
[0110] The silicase therefore exhibits the properties as necessary,
in order to eliminate silicon dioxide from the lung and/or to
modulate the progression of this lung disease.
[0111] Different methods fort he administration of the recombinant
enzyme can be taken into account: a) as enzyme preparation, b)
packaged in liposomes, c) associated with microspheres or d)
adenovirus-mediated gene transfer.
[0112] Microspheres as carrier-systems for the recombinant silicase
for the treatment of silicosis (dissolution of SiO.sub.2) e.g. can
be prepared from sponge-collagen in analogy to
calf-collagen-microspheres (Rossler et al., Pharmazie 49 (1994)
175-179). Die sponge-collagen-microparticles are loaded by
adsorption of the recombinant protein (silicase), as described
(Rossler et al., J. Microencapsulation 12 (1995) 49-57; Berthold et
al., Eur. J. Pharm. Biopharm. 45 (1998) 23-29). The advantages of
collagen are its bio-degradability as well as its low toxicity and
immunogenicity. As further "Delivery"-systems, amongst other,
liposomes with the encased recombinant enzyme as well as
lipid-nanoparticle can be taken into account (Jenning et al., Eur.
J. Pharm. Biopharm. 49 (2000) 211-218).
8.3. Modification of the Properties of Cells by Transfection with a
Silicase Gene/cDNA-Containing Plasmid
[0113] Through a transfection of cells with a silicase
gene/cDNA-containing plasmid, their properties can be modified,
allowing for, amongst others, a use in the production of bone
replacement materials or a gene therapy (e.g. in silicosis).
EXPLANATION TO THE FIGURES
[0114] In the following, the explanatory legends for the
accompanying drawings and the sequence protocol are given. It
shows:
[0115] SEQ ID No. 1: The amino acid sequence of the silicase
according to the invention invention from S. domuncula
(SIA_SUBDO)
[0116] SEQ ID No. 2: The nucleic acid sequence of the cDNA of the
silicase according to the invention from S. domuncula.
[0117] FIG. 1:
[0118] Identification of transcripts in Primmorphs that were
up-regulated after incubation in 60 .mu.M silicon for 1 or 3 days,
with the aid of the technique of the differential display. The
Primmorphs were either maintained at the normal
silicon-concentration of 5 .mu.M (lane a) or were incubated in the
presence of 60 .mu.M silicon (lane b and c). The RNA was extracted
and used for the analysis. For amplification of the transcripts two
different random primer (1 and 2) were used. Those transcripts are
marked (>) which only occurred at higher silicon-concentration
(lane b and c) and were analyzed.
[0119] FIG. 2:
[0120] top: Amino acid sequence derived from the nucleotide
sequence of the open reading frame (coding region) of the S.
domuncula silicase-cDNA (SIA_SUBDO). bottom: Nucleotide sequence of
the S. domuncula Silicase-cDNA (SIA_SUBDO). The amino acid sequence
derived from the nucleotide sequence of the open reading frame is
given below the nucleotide sequence.
[0121] FIG. 3:
[0122] (A) Alignment of the S. domuncula silicase (SIA_SUBDO) with
the human carbonic anhydrase 11 (carbonate dehydratase II)
(CAH2_HUMAN; P00918). The carbonic anhydrase domain is framed (
=e-CAdom= ). The characteristic amino acids that form the
eukaryotic-type-carbonic anhydrase-signature, are marked
(.tangle-solidup.: found in both sequences; .box-solid.: present
only in the carbonic anhydrase but not in the silicase). The
additional symbols (+) indicate those residues, that form the
hydrogen-network of the active center. The three zinc-binding
histidine-residues are marked (Z). Similar amino acid residues
between both sequences are highlighted. The borders of the long
(.about.rec.about. to .about.rec.about.) as well as the short
recombinant silicase (.about.rec-s.about. to .about.rec.about.) are
marked and underlined twice. (B) phylogenetic tree, constructed
with the sponge-silicase and following related enzymes: human
carbonanhydrase I (carbonate dehydratase I) (CAH1_HUMAN; P00915),
II (CAH2.about.HUMAN), III (CAH3_HUMAN; P07451), IV (CAH4_HUMAN;
P22748), VI (CAH6_HUMAN; P23280), VII (CAH7_HUMAN; P43166), VIII
(CAH8_HUMAN; P35219), IX (CAH9_HUMAN; Q16790), X (CAHA HUMAN;
Q9NS85), VA (CAH5_HUMAN; P35218), VB (CA5B_HUMAN; Q9Y2D0), XII
(CAHC_HUMAN; 043570), XIV (CAHE_HUMAN; Q9ULX7), carbonic anhydrase
of Caenorhabditis elegans (CAH_CAEEL; Nu-510674.1), carbonic
anhydrase of Drosophila melanogaster (CAH1_DROME; NP523561.1),
carbonic anhydrase of the plants Arabidopsis thaliana (CAH-I_ARATH;
NP.sub.--196038.1) and Chlamydomonas reinhardtii (carbonate
dehydratase 1) (CAH1CHLRE; P20507) as well as bacterial carbonic
anhydrases from Neisseria gonorrhoeae (CAH_NEIGO; Q50940),
Klebsiella pneumoniae (CAH_KLEPN; 052535) and the cyanobacteria
Nostoc sp. PCC 7120 (CAHANASP; P94170). The latter sequence were
used as outgroup. The measure bars indicate an evolutionary
distance of 0.1 amino acid-substitutions per position in the
sequence. The phylogenetic tree was constructed by means of
"Neighbor-Joining" ("Neighbor" program: Felsenstein, J. (1993).
PHYLIP, ver. 3.5. University of Washington, Seattle).
[0123] FIG. 4:
[0124] Production of the recombinant silicase. The recombinant S.
domuncula Silicase (rSIA_SUBDO) was produced as GST-fusion protein.
The long as well as the short SDSIA were cloned in a
pGEX-4T-2-plasmid that contained the glutathione-S-transferase
(GST)-gene. The fusion proteins were isolated either without prior
induction with IPTG (-IPTG) or after incubation with IPTG (+IPTG)
for 4 or 6 hours, subsequently cleaved, purified and subsequently
subjected to the Na-DodSO.sub.4-PAGE. The gel was stained with
Coomassie Brillant Blue. The purified long form rSIA_SUBDO-I with a
size of 43 kDa as well as the short form (M.sub.r 32 kDa) of the
silicase were obtained.
[0125] FIG. 5:
[0126] Determination of the enzymatic activity of the silicase in
the carbonic anhydrase and in the silicase assay. The recombinant
silicase was added to the reaction mixtures, in concentrations
between 1 and 10 .mu.g per assay (0.56 .mu.l). For the
determination of the carbonic anhydrase-activity (.box-solid.)
p-nitrophenyl acetate was used as a substrate. The released
p-nitrophenol was measured at a wavelength von 348 nm. The activity
of the silicase (.circle-solid.) was determined with the use of S.
domuncula spicules. The released silicic acid as formed by
depolymerisation (decomposition) of amorphous silicon dioxide was
determined with the aid of the "Silicon Test" colorimetric
reaction.
[0127] FIG. 6:
[0128] Effect von silicon on the formation of spicules in
Primmorphs. For the formation of the Primmorphs dissociated cells
of the marine sponge S. domuncula were incubated in sea water,
supplemented with 10% RPM 11640-Medium and 30 .mu.M Fe(+++). The
Primmorphs were then transferred for 3 days into a medium (RPMI
1640, Fe (+++)) that was enriched with 60 .mu.M silicon. (A) The
Primmorphs were incubated in medium plus silicon. magnification:
.times.6. (B) In some cases the Primmorphs started with the
synthesis of spicules (sp). magnification: .times.10. For the
semi-quantitative determination, the Primmorphs were pressed
between two cover slides (C and D). (C) Primmorphs that were
incubated in the absence of silicon inkubiert were nearly
completely without spicules, whereas those that were cultivated in
the presence of silicon contained newly formed spicules (>);
(D); magnification: .times.200
[0129] FIG. 7:
[0130] Expression of the silicase, the silicatein, and the
isocitrate-dehydrogenase, determined by Northern-Blotting. The RNA
was extracted from Primmorphs that were incubated for 1 to 3 days
in the absence of additional silicon (-Si) or in the presence of 60
.mu.M silicon (+Si). 5 .mu.g of the total RNA were
electrophoretically separated, blotted onto Nylon membranes and
hybridized with the following probes: SDSIA (silicase), SDSILICA
(silicatein), and SDIDH (.alpha.-subunit of the
isocitrate-dehydrogenase). The sizes of the transcripts are
given.
[0131] FIG. 8:
[0132] Enzymatic reactions as mediates by the silicase (carbonic
anhydrase) of S. domuncula. In [1] the conversion of CO.sub.2 into
HCO.sub.3.sup.- is shown. In [2], the reaction of the silicase is
shown. The silicase binds one zinc atom with its three
histidine-residues. The zinc ion, a Lewis-acid, binds a
hydroxide-anion that is derived from water, a Lewis-base. The
silicase/zinc-complex undertakes a nucleophilic attack auf a
silicon atom between the oxygen bonds. Thereby, the hydrolysis of
the polymeric silicon dioxide is achieved, which first--with one of
both product halves--maintains bound to the enzyme. Upon further
consumption of H.sub.2O, the product is released until finally free
silicic acid is left after several cycles.
[0133] FIG. 9:
[0134] Left: Spicules (needles) of Suberites domuncula after 6 hour
incubation in the absence of silicase. Right: Spicules of Suberites
domuncula after 6 hour incubation in the presence of silicase. The
incubation took place under the conditions as described in table 2.
Sequence CWU 1
1
2 1 379 PRT Suberites domuncula 1 Met Ser Ala Ile Leu Lys Arg Asn
Val Pro Ile Gln Arg Val Gly Leu 1 5 10 15 Pro Leu Thr Ser Tyr Val
Ser Arg Trp Ala Ser Ala Leu Pro Thr Arg 20 25 30 Thr His Pro Phe
Tyr Lys Leu Val Asp Asp Ser Thr Thr Pro Val Thr 35 40 45 Arg Ser
Thr Leu Leu Ser Ala His Met Val Asp Thr Leu Leu Asp Glu 50 55 60
Asn Gln Gln Ser Arg His Glu Asn Gln His Thr Asp Thr Ser Tyr Lys 65
70 75 80 Met Tyr Gln Gly Leu Lys Phe Val Val Lys Thr Leu Phe Thr
Pro Ser 85 90 95 Lys Cys His Arg His Phe Ser Thr Ser Ala His Leu
Ser Ala Met Gly 100 105 110 Arg His Gln Ser Pro Ile Asn Ile Ile Thr
Ser Ser Thr Thr Lys Gly 115 120 125 Pro Ser Leu Lys Pro Leu Lys Phe
Ser Lys Ser Trp Asp Lys Pro Val 130 135 140 Ile Gly Thr Val Lys Asp
Thr Gly Tyr Tyr Leu Lys Phe Ala Pro Glu 145 150 155 160 Ser Ala Ala
Glu Lys Cys Thr Leu His Thr Tyr Asn Gly Glu Tyr Ile 165 170 175 Leu
Asp His Phe His Tyr His Trp Gly Lys Lys Asp Gly Glu Gly Ala 180 185
190 Glu His Phe Ile Asp Gly Lys Gln Tyr Asp Ile Glu Phe His Phe Val
195 200 205 His Lys Lys Val Gly Leu Thr Asp Pro Asp Ala Arg Asp Ala
Phe Ala 210 215 220 Val Leu Gly Val Phe Gly Lys Ala Asp Pro Arg Leu
Lys Ile Asn Gly 225 230 235 240 Ile Trp Glu Leu Leu Ser Pro Ser Thr
Val Leu Thr Val Asp Ser Thr 245 250 255 Arg Asn Val Ala Asp Val Val
Pro Ser Lys Leu Leu Pro Ser Ala Arg 260 265 270 Asp Tyr Phe His Tyr
Glu Gly Ser Leu Thr Thr Pro Thr Tyr Gly Glu 275 280 285 Val Val His
Trp Phe Val Leu Asn Glu Pro Ile Ala Val Pro Ser Glu 290 295 300 Tyr
Leu Ser Ala Leu Arg Gln Met Gln Ala Asp Lys Glu Gly Thr Val 305 310
315 320 Ile Asp Ser Asn Tyr Arg Glu Leu Gln Glu Val His Asn Arg Pro
Val 325 330 335 Gln Arg Phe Lys Ser Asp Glu Gln Gly Arg Gly Glu Phe
Asp Asp Ile 340 345 350 Ser Lys Asn Glu Asp Ile Val Glu Asp Leu Ser
Lys Leu Ser Gly Asn 355 360 365 Phe Ile Arg Glu Leu Val Arg Lys Ile
Tyr Trp 370 375 2 1396 DNA Suberites domuncula 2 gaattcggca
cgagggacaa ctttgcataa cttttactgt ccatgtttaa cgtttagatc 60
tagtactagt agtctacaag aacaactgtc aacaactgtc agattatgtg tataaaccaa
120 gatgtctgca attcttaaga gaaacgtacc tatccaaaga gtcggtctcc
cactgacctc 180 ctatgtcagt agatgggctt ctgctctgcc caccaggacc
catccttttt acaagttggt 240 tgatgacagt accaccccag tgacaaggtc
tactcttctc agtgctcata tggttgacac 300 cttgctagat gagaaccagc
agagcagaca tgaaaaccaa cacacagaca cgtcttacaa 360 aatgtaccag
ggattaaaat ttgttgtaaa gacgctgttt actccatcga aatgccaccg 420
tcacttctcc acatcagctc atttgtctgc catgggtcga catcaatccc ccatcaatat
480 aatcacctcc agtacgacca aaggaccgtc attgaaaccg ttaaaattta
gcaagagttg 540 ggacaagcca gtaatcggca ccgtcaaaga tactggctat
tatcttaaat ttgcaccaga 600 atctgcagca gagaagtgca cattgcatac
gtacaatggt gaatatatcc tagatcattt 660 ccattatcac tgggggaaga
aggatgggga aggagcagag catttcatcg atggaaaaca 720 atacgacatc
gagttccact ttgtacataa aaaggttggg ttgactgatc cagatgctag 780
agacgctttt gctgttttgg gcgtttttgg aaaggccgac cctcgtttga agatcaatgg
840 aatctgggag ctactctcac cgtcaactgt cctgactgtc gactcaacac
gaaacgtcgc 900 tgatgttgtt ccctctaagc ttctcccaag tgccagagac
tattttcact atgaaggttc 960 tttgaccaca cctacgtatg gtgaggttgt
gcactggttt gttctcaatg aacccatagc 1020 tgtccctagt gagtatctgt
cagctctgag acagatgcaa gctgacaaag aaggtactgt 1080 gattgactca
aactatcgag agcttcaaga agtccacaat cgacctgtgc aacgatttaa 1140
gagtgatgag caagggagag gagaatttga cgatatttct aagaatgagg acattgtgga
1200 ggacttgtct aaattgtctg gtaactttat tagagagctg gtcaggaaga
tatattggtg 1260 acctttttct acacttgtta gagttttagg ccagaataca
tttcatcatt tggactgtta 1320 ttttgtgtac actgcttagc agtttatata
aacactacaa tgccattatt ataatatagc 1380 caatgctgtg atttga 1396
* * * * *