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INTRODUCTION |
Silicon oxide minerals, the main constituents of the earth's
crust, are not exclusively formed by geological processes. In fact,
hydrated silicon dioxide (silica), the second most abundant biogenic
mineral (biomineral), is produced by a wide range of organisms
including animals and higher plants (1). A large proportion of biogenic
silica is formed by diatoms (2), which are unicellular algae that are
ubiquitously present in marine and freshwater habitats (3). The main
attribute of a diatom cell is its silica based cell wall. The intricate
and ornate silicified cell walls of diatoms are one of the most
outstanding examples of nanoscale-structured materials in nature. In
the past, diatoms have been studied as model organisms to investigate
the biochemical basis of biological silica formation (4-6). This has
led to the discovery of silicic acid transporter proteins (7, 8) and unique organic components that are associated with biosilica
(9-11).
Interest in silica biomineralization has been greatly increased by the
recognition that the organic molecules that mediate the formation of
silica structures in vivo could be useful tools in materials
technology for biomimetic production of nanostructured silica in
vitro (12-14). Recently, silica-associated components from
different diatom species were identified that mediate the formation of
silica nanospheres in vitro from a silicic acid solution (10, 11). These components are long-chain polyamines and polycationic polypeptides termed silaffins. The chemical structures of the polyamines have been completely elucidated. They are composed of linear
chains of 8 to 20 N-methylated propylamine units that are
attached to putrescine or a putrescine derivative (11). In contrast,
there is only incomplete information about the chemical structure of
the silaffins. Recently, a silaffin-encoding gene, termed
sil1, has been cloned from the diatom Cylindrotheca
fusiformis. The encoded polypeptide sil1p serves as a precursor
molecule, which becomes proteolytically processed and
post-translationally modified to produce the silica-precipitating
peptides silaffin-1A and silaffin-1B. It has been demonstrated that
silaffin-1A represents a mixture of peptide isoforms, and that their
silica-precipitating activity depends on the presence of modified
lysine residues (10). So far, two types of modified lysine residues
(denoted Lysx and Lysy) have been characterized
from the N-terminal octapeptide fragment SSKxKySGSY
that is common to all silaffin-1A isoforms. Lysx represents a
lysine residue that carries on its
-amino group a linear polyamine
consisting of 5 to 11 N-methylated propylamine units.
Lysy has been shown to represent
-N,N-dimethyllysine. However, no information was
available for the chemical structures of the remaining parts of the
silaffin-1A isoforms. In the present study we describe the
silica-precipitating properties and complete the chemical structures of
the peptides silaffin-1A1 and silaffin-1A2,
which together account for all peptide isoforms of silaffin-1A.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Synthetic
-N,N,N-trimethyl-
-hydroxylysine (19) was a gift from
B. E. Volcani (Scripps Institute for Oceanography, University of
California, San Diego, La Jolla, CA).
Culture Conditions--
Cylindrotheca fusiformis
was grown in artificial sea water medium as described
previously (15).
Isolation of silaffin-1A1 and
silaffin-1A2--
The silaffin-1A fraction was isolated
from purified cell walls of C. fusiformis as
described previously (10) and subjected to high pressure liquid
chromatography (HPLC)1 on a
Sephasil C18 2.1/10 column using the SMART-System (Amersham Pharmacia Biotech). Elution of peptides was performed by increasing the
concentration of buffer B from 0 to 25% in 35 min (buffer A: 0.1%
trifluoroacetic acid in H2O; buffer B: 0.085%
trifluoroacetic acid in acetonitrile). Fractions containing
silaffin-1A1 and silaffin-1A2, respectively,
were lyophilized and the residues were dissolved in
H2O.
Digestion of Silaffin-1A1 with Chymotrypsin and
Separation of Peptides--
Silaffin-1A1 (180 µg) was
dissolved in 100 µl of 50 mM Tris/HCl, pH 8, and 9 µg
of chymotrypsin
(N
-p-tosyl-L-lysine
chloromethyl ketone-treated; Sigma) was added. Incubation was at
37 °C for 15 h. The resulting chymotryptic peptides were
separated by HPLC using the same conditions as described above.
Acid Hydrolysis and Hydrazinolysis--
Complete degradation of
silaffins to yield free amino acids was performed by gas phase
hydrolysis using 6 M HCl at 110 °C for 6-16 h. For
hydrazinolysis anhydrous hydrazine was prepared from hydrazonium
cyanurate (Fluka) according to the method of Nachbaur and Leiseder
(16). Silaffin peptides were lyophilized in glass reaction tubes
(ReactiVial, Pierce), and 100 µl anhydrous hydrazine was added to
each sample under a nitrogen atmosphere. The reaction tubes were sealed
tightly and incubated at 110 °C for 24 h. Subsequently,
hydrazine was evaporated by lyophylization, and the residues were
dissolved in 50 mM ammonium acetate.
Alkylation of Modified Lysine Residues--
A dried acid
hydrolysate of silaffin-1A was dissolved in 50 mM sodium
phosphate, pH 7, and subjected to reductive ethylation with sodium
cyanoborohydride and acetaldehyde according to a previously described
protocol (17).
Amino Acid Sequencing--
Peptides were sequenced by automated
Edman degradation on a Procise 492A sequencer (PE Biosystems) with
on-line detection of the phenylthiohydantoine amino acids
according to the manufacturer's instructions.
Mass Spectrometry--
Electrospray ionization mass spectrometry
(ESI-MS) and fragmentation analysis were performed using an Ion Trap
ESQUIRE LC (Bruker) instrument. Samples were infused by a nanospray
source in either 1 mM ammonium acetate, 50%
CH3CN (for analysis of polyamine modified lysine residues)
or 0.5% acetic acid, 50% methanol (for analysis of peptides and amino acids).
Determination of Peptide Concentration and Silica Precipitation
Assay--
These procedures were performed as described
previously (10).
Electron Microscopy--
Silica precipitates were washed with
H2O, mounted onto a graphite coated coverslip, and analyzed
on a LEO1530 field-emission scanning electron microscope.
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RESULTS |
Isolation and Sequence Analysis of Silaffin-1A Isoforms--
Amino
acid sequencing of silaffin-1A exhibited the N-terminal sequence
SSKxKySGSYSG(S/Y) (10), indicating the existence of at
least two different but related peptide species within the silaffin-1A
preparation. Reversed phase HPLC on a C18 column produced
two peptide fractions, which were termed silaffin-1A1 and
silaffin-1A2, respectively (Fig.
1A). Amino acid sequencing
resulted in the sequence SSKxKySGSYSGSKzGS for
silaffin-1A1 (Lysx and Lysy were previously
shown to represent modified lysine residues; the chemical nature of
Lysz will be described below) and the sequence
SSKxKySGSYSGYSTKxKySGS for
silaffin-1A2. No further amino acid signals were observed after sequencing cycle 14 and 18, respectively. Mass spectrometry indicated that both of these fractions contain peptide isoforms differing by increments of 71 mass units (Table
I). This mass heterogeneity can be
explained precisely by the variation in chain length (number of
methylated propylamine units) of the polyamine modification present on
lysine derivative Lysx. A comparison of the
silaffin-1A1 and silaffin-1A2 sequences with
the sequence of the silaffin precursor polypeptide sil1p clearly
demonstrates that the silaffin-1A1 peptides are derived from any of the repeats R3-R7 of sil1p, whereas
silaffin-1A2 is derived from repeat R2 (Fig.
1B).

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Fig. 1.
Analysis of silaffin-1A isoforms.
A, separation of silaffin-1A1 and
silaffin-1A2 on a reversed phase C18 column.
B, schematic structure of the silaffin precursor polypeptide
sil1p. The black pentagons denote the repeating unit
elements from which the silaffins are generated. The amino acid
sequences of repeating units R2 and R3-R7, respectively, are
bracketed. Repeating unit R1 gives rise to silaffin-1B (10).
The white bar denotes the signal peptide, and the gray
oval indicates the highly acidic prosequence (10).
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Table I
Molecular masses of silaffin-1A1 and silaffin-1A2
The (m + H)+ masses of peptides as determined
by ESI-MS and as calculated according to the chemical structures (see
Fig. 5) are compared. The values for n + m
indicate the total number of propylamine units attached to respective
lysine residues. The column denoted indicates the mass differences
between neighboring peptides.
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The ratio of peak areas of silaffin-1A1 and
silaffin-1A2 in the HPLC chromatogram is about 5 to 1. Therefore, it is reasonable to assume that each of the repeats R3-R7
of sil1p contributes to silaffin-1A1 production (Fig.
1B). A comparison of the sil1p sequence (10) with the
results from amino acid sequencing of isolated silaffins reveals the
following structural features of silaffin-1A1 and
silaffin-1A2. 1) All of the lysine residues present are
post-translationally modified. Three types of modified lysine residues
(designated Lysx, Lysy, and Lysz) can be
distinguished in silaffin-1A1. Lysy produces a
signal between Arg and Tyr in the chromatogram of the amino acid
sequencer and was previously identified as
-N,N-dimethyllysine (10). Lysz is a so far
unidentified lysine derivative exhibiting a characteristic peak between
Ala and Arg. Lysx denotes a lysine derivative that does not
show up at all in automated amino acid sequencing. For lysine
derivative Lysx at position 4, a long-chain polyamine was
previously shown to be attached to the
-amino group (10). 2) Neither
the C-terminal amino acid sequence RRIL predicted by the gene sequence
of repeat R2 nor the corresponding sequences KRRNL and KRRIL predicted
from repeats R3 to R7 showed up in amino acid sequencing of
silaffin-1A2 and silaffin-1A1, respectively.
Therefore, these residues may either have become removed or
post-translationally modified during processing of the silaffin
precursor polypeptide.
Complete Amino Acid Sequence of Silaffin-1A1--
To
further analyze the chemical structure, silaffin-1A1 was
digested with chymotrypsin, which generates the previously described N-terminal octapeptide SSKxKySGSY (see the
Introduction) and the C-terminal fragment SGSKzGS (Fig.
2). In reversed phase C18
HPLC, the N-terminal octapeptide separates into five fractions (Fig.
2), which differ in masses by multiples of 71 Da (Table
II) due to heterogeneity with respect to
the chain length of the polyamine modification (10). Surprisingly, the
same mass differences were observed in the subfractions derived from
the C-terminal peptide SGSK3GS (Table II), suggesting that
this peptide also contains a lysine residue carrying the polyamine
modification. Because this type of lysine derivative is not detectable
by automated amino acid sequencing, we hypothesized that it may
constitute the C terminus, and therefore the sequence of the C-terminal
peptide may rather be represented by
SGSKzGSKx. To investigate
this, the material from fraction 3 (C-terminal peptide) and
fraction 8 (N-terminal octapeptide), respectively, was subjected to
hydrazinolysis, and the resulting products were analyzed by ESI-MS.
Hydrazinolysis leads to the breakdown of the peptide backbone, and all
amino acids residues originally placed within the polypeptide chain
become converted to the corresponding hydrazides. Only the C-terminal
residue is released as free amino acid (18). As expected,
hydrazinolysis of the material from fraction 8 (N-terminal octapeptide)
generated a molecule of mass (m + H)+ = 729.8 Da
(Fig. 3A). This molecule
corresponds to the hydrazide of a lysine derivative carrying eight
methylated propylamine units (the molecular mass of the hydrazide is 14 Da higher compared with the free amino acid derivative). In contrast, a
molecule of mass (m + H)+ = 715.8 Da was found
among the hydrazinolysis products from fraction 3 (C-terminal peptide)
indicating that a lysine residue carrying eight methylated propylamine
units is indeed present at the C terminus of the original peptide (Fig.
3B). This result demonstrates that the correct peptide
sequence of silaffin-1A1 is represented by
SSKxKySGSYSGSKzGSKx.

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Fig. 2.
Analysis of silaffin-1A1
fragments. Silaffin-1A1 was digested with
chymotrypsin, and the resulting peptides were separated on a reversed
phase C18 column. The amino acid sequences of the
C-terminal peptides (fractions 1-5) and the N-terminal peptides
(fractions 6-10) as determined by complete amino acid sequencing are
indicated.
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Table II
Molecular masses of chymotryptic peptides from silaffin-1A1
Peptides were separated by reversed phase HPLC and individual fractions
(see Fig. 2 for numbering) were analyzed by ESI-MS. The
(m + H)+ masses of peptides are shown in the
middle column. The column denoted indicates the mass differences
between neighboring peptides.
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Fig. 3.
Determination of the C-terminal amino acid of
silaffin-1A1. Chymotryptic peptides 3 and 8 (see Fig.
2 for numbering) generated from silaffin-1A1 were subjected
to hydrazinolysis, and the products were analyzed by ESI-MS. Only the
m/z regions corresponding to the singly charged
ions of lysine derivative Lysx are shown.
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Identification of
-N,N,N-Trimethyl-
-hydroxylysine--
To
identify the chemical structure of lysine derivative Lysz,
complete acid hydrolysis of silaffin-1A1 was performed, and
the masses of the resulting products were analyzed by ESI-MS (data not
shown). Apart from the masses of the known amino acid constituents of
silaffin-1A1 (Ser, Gly, Tyr, Lysx, and
Lysy) a molecule of mass (m + H)+ = 205.1 was detected. This molecule was present only in the acid hydrolysate of the C-terminal peptide of silaffin-1A1 (data
not shown), indicating that (m + H)+ = 205.1 corresponds to the molecular mass of the lysine derivative Lysz. The (m + H)+ = 205.1 ion was
isolated using the ion trap mode of the mass spectrometer and subjected
to collision induced fragmentation. The elimination of 59 and 18 mass
units indicated the presence of a trimethylammonium group as well as an
hydroxy group within this lysine derivative. Indeed, the obtained
product ion spectrum (Fig. 4A)
matches the spectrum obtained from authentic
-N,N,N-trimethyl-
-hydroxylysine (Fig. 4B).
This lysine derivative had previously been found in acid hydrolysates
obtained from total cell wall preparations of different diatom species
including C. fusiformis (19).

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Fig. 4.
Identification of
-N,N,N-trimethyl- -hydroxylysine.
Product ion spectra obtained by collision-induced fragmentation.
A, product ions obtained from the (m + H)+ = 205.1 Da ion that is present in the amino acid
hydrolysate of silaffin-1A1. B, product ion
spectrum obtained from authentic
-N,N,N-trimethyl- -hydroxylysine.
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Chemical Structures of Silaffin-1A1 and
Silaffin-1A2--
Taken together, the data of
silaffin-1A1 analysis lead to the structural model
presented in Fig. 5. This model exactly
explains the molecular masses of all peptide isoforms found to be
present within the silaffin-1A1 fraction (Table
I).

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Fig. 5.
Chemical structures of
silaffin-1A1 and silaffin-1A2. The
polypeptide backbones are depicted by the one-letter amino acid code.
The chemical structures of the side chains of only the modified lysine
residues are shown.
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The molecular masses of the silaffin-1A2 peptide isoforms
(Table I) are consistent with the assumption that both the lysine derivatives Lysx at positions 3 and 14 carry the polyamine
modification and that the lysine derivatives Lysy found at
positions 4 and 15 are
-N,N-dimethyllysine. This was confirmed by acid hydrolysis of silaffin-1A2 and subsequent
ESI-MS analysis.
-N,N-Dimethyllysine and
polyamine-modified lysine derivatives were the only modified amino
acids present in this hydrolysate. The structural model for
silaffin-1A2 is shown in Fig. 5.
Methylation Pattern of the Long-chain Polyamine
Modification--
Structural analysis of long-chain polyamines by mass
spectrometry revealed that their collision-induced fragmentation is
caused exclusively by the cleavage of C-N bonds (11). According to this finding, the previously proposed structure of the polyamine moiety
linked to lysine derivatives Lysx in silaffin-1A (10) has to be
reconsidered. A shift of two methyl groups within the polyamine chain
enables the interpretation of all the fragment ions observed (10) by
allowing C-N bond cleavages only. Therefore, a modified structural
model is proposed for lysine derivative Lysx (included in Fig.
5) in which the polyamine moiety is dimethylated at its terminal amino
group, thus representing a methylation isomer of the previously
proposed structure. The modified structural model was confirmed by
reductive ethylation of lysines Lysx and fragmentation analysis
(by mass spectrometry) of the resulting ethylated derivatives.
Reductive ethylation introduced exactly four ethyl groups into each
Lysx isoform, irrespective of chain-length variations in the
polyamine moiety (data not shown). The fragment ion spectrum of one of
these ethylated derivatives (Fig.
6A) demonstrates that the
terminal amino group of the polyamine moiety is dimethylated, because
it is only the
-amino group of the lysine core as well as the amino group of the very first propylamine unit that can be converted to an
N-ethyl derivative (in addition to the
-amino group of the lysine core; Fig. 6B). This fact clearly indicates that
both of these amino groups exist as secondary amines in the parent molecule, i.e. they are not methylated (these amino groups
were assumed to be methylated in the previous structural model).

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Fig. 6.
Structural analysis by ethylation of lysine
derivative Lys1. Ethylated lysine residue Lysx
of molecular mass (m + H)+ = 898.9 Da (molecular
mass before ethylation was (m + H)+ = 786.9 Da,
indicating the introduction of four ethyl groups) was isolated by the
ion trap mode of the mass spectrometer and subjected to
collision-induced fragmentation. A, product ion spectrum
after fragmentation. B, proposed structure (schematic) of
the (m + H)+ = 898.9 Da ion. Cleavage positions
that lead to the observed fragment ion spectrum are depicted by
arrows, and the corresponding molecular masses are
indicated.
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Silica Precipitation--
It was previously shown that the silica
precipitation activity of silaffin-1A peptides at pH values < 7 is dependent on the lysine modifications (10). Because the presence of
-N,N,N-trimethyl-
-hydroxylysine clearly distinguishes
silaffin-1A1 from silaffin-1A2 (Fig. 5), it was
investigated as to whether the two silaffin-1A isoforms have different
pH-dependent silica-precipitating properties. In an
in vitro assay, the amount of silica precipitated by
silaffin-1A1 and silaffin-1A2, respectively,
was found to be fairly constant at different pH values and almost
identical for silaffin-1A1 (9.0-11.9 nmol of Si/nmol of
peptide) and silaffin-1A2 (10.3-11.7 nmol of Si/nmol of
peptide). Only at pH 5 did silaffin-1A1 show a slightly lower silica-precipitating activity as compared with
silaffin-1A2 (Fig.
7A). The structures of the
silica precipitates were analyzed by scanning electron microscopy. At
all pH values silaffin-1A peptides induced the formation of spherical
silica nanoparticles, but at the level of scanning electron microscope
resolution, no morphological difference was noted between the
precipitates induced by silaffin-1A1 (Fig. 7B)
and silaffin-1A2 (Fig. 7C).

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Fig. 7.
Silica precipitation by
silaffin-1A1 and silaffin-1A2.
A, pH dependence of peptide-induced silica precipitation.
The solid line (measured values in squares) shows
the result for silaffin-1A1 and the dotted line
(measured values in triangles) represents the results for
silaffin-1A2. B and C, scanning
electron microscopic images of silica precipitates induced at pH 6.4 by
3 mg/ml silaffin-1A1 (B) and 3 mg/ml
silaffin-1A2 (C).
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DISCUSSION |
The present study describes for the first time the complete
chemical structures of silica-precipitating peptides found in cell
walls of the diatom C. fusiformis. These are
silaffin-1A1 and silaffin-1A2, which consist of
15 and 18 amino acid residues, respectively. Both peptides contain a
total of four lysine residues, and all of these are targets for
post-translational modifications. In silaffin-1A2, the
lysine residues are clustered in two pairs with the first residue being
linked to a long-chain polyamine and the second lysine being converted
to
-N,N-dimethyllysine. In silaffin-1A1, the
same type of modified lysine pair is present only once within the
N-terminal part of the peptide. The remaining two lysine residues in
the C-terminal part are separated by two intercalated amino acids; this
motif appears to alter the strategy of post-translational modification.
The lysine residue at position 12 becomes modified to
-N,N,N-trimethyl-
-hydroxylysine, and it is now the
C-terminal lysine residue that carries a long-chain polyamine
modification. Remarkably, more than 30 years ago, Nakajima and Volcani
(19) isolated and characterized for the first time
-N,N,N-trimethyl-
-hydroxylysine in acid hydrolysates
of total cell wall preparations from a number of diatoms. However, the corresponding proteins in diatoms containing this special type of
modification remained elusive. Silaffin-1A1 is (to our
knowledge) the first polypeptide found in nature containing the
-N,N,N-trimethyl-
-hydroxylysine residue.
Despite the structural differences of silaffin-1A1 and
silaffin-1A2, both polycationic peptides show almost
identical silica-precipitating activities and promote the formation of
silica nanospheres in vitro (see Fig. 7). This result
suggests that the silica-precipitating activities of
silaffin-1A1 and silaffin-1A2 are dependent
mainly on the polyamine modification attached to lysine residues. This is consistent with the finding that long-chain polyamines attached to
putrescine that were isolated from diatom cell walls are also able to precipitate silica nanospheres (11), whereas synthetic silaffin
peptides lacking the lysine modifications are unable to precipitate
silica at pH < 7 in vitro (10). In this respect it is
interesting to note that silica formation in diatoms takes place in an
acidic, intracellular compartment (20), and thus the polyamine moieties
of the silaffin-1A peptides appear to be essential to mediate silica
precipitation under physiological conditions. The
-N,N,N-trimethyl-
-hydroxylysine present in
silaffin-1A1 is a structural element that might influence
the ultrastructure of the precipitating silica. Remarkably, quaternary
ammonium compounds are used in the technical production of zeolites for
patterning of silicate structures in the nanometer size range (21).
Possibly, the
-N,N,N-trimethyl-
-hydroxylysine residue
exerts a similar function in biosilica formation.
The role of the polypeptide backbones in silaffin-1A-mediated silica
formation is much less clear. Isolation of silaffins from diatom
biosilica involves treatment with anhydrous hydrogen fluoride that
converts silica to volatile silicon tetrafluoride. Although this
treatment does not attack peptide bonds, it does however specifically
cleave O-glycosidic bonds (22). Silaffins contain a large
number of hydroxyamino acid residues, which may be targets for
O-glycosylation. However, a completely different technique
for the extraction of silaffins from biosilica is required to
investigate this possibility.
Comparison of the silaffin-1A1 and silaffin-1A2
sequences with the sequences deduced from the sil1 gene
revealed that during maturation of the silaffins, the C-terminal
tetrapeptides RRIL and RRNL, respectively, become cleaved off. This
processing step completely removes all arginine residues that are
originally present in the silaffin precursor polypeptide sil1p (see
Fig. 1B). Remarkably, arginine is the biosynthetic precursor
of putrescine (23), and the latter has been shown to serve as the
attachment site for long-chain polyamines in C. fusiformis
and other diatoms (11). Therefore, it is intriguing to speculate
that sil1p is also the precursor for the putrescine-linked polyamines.
After conversion of the arginine residues in the silaffin precursor to
ornithine residues, the latter may become modified by the same
enzymatic machinery that attaches propylamine units to the appropriate
lysine residues in silaffins. Subsequently, silaffin peptides and
putrescine-based polyamines could be produced simultaneously by
proteolytic processing and decarboxylation of the polyamine-modified
ornithine residues. If so, sil1p of C. fusiformis would give
rise to two different sets of silica-precipitating molecular species.