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Silica-precipitating Peptides from Diatoms

2001; Elsevier BV; Volume: 276; Issue: 28 Linguagem: Inglês

10.1074/jbc.m102093200

1083-351X

Nils Kröger, Rainer Deutzmann, Manfred Sumper,

Advanced Proteomics Techniques and Applications

Two silica-precipitating peptides, silaffin-1A1 and-1A2, both encoded by thesil1 gene from the diatom Cylindrotheca fusiformis, were extracted from cell walls and purified to homogeneity. The chemical structures were determined by protein chemical methods combined with mass spectrometry. Silaffin-1A1 and -1A2 consist of 15 and 18 amino acid residues, respectively. Each peptide contains a total of four lysine residues, which are all found to be post-translationally modified. In silaffin-1A2 the lysine residues are clustered in two pairs in which the ε-amino group of the first residue is linked to a linear polyamine consisting of 5 to 11N-methylated propylamine units, whereas the second lysine is converted to ε-N,N-dimethyllysine. Silaffin-1A1 contains only a single lysine pair exhibiting the same structural features. One of the two remaining lysine residues was identified as ε-N,N,N-trimethyl-δ-hydroxylysine, a lysine derivative containing a quaternary ammonium group. The fourth lysine residue again is linked to a long-chain polyamine. Silaffin-1A1 is the first peptide shown to contain ε-N,N,N-trimethyl-δ-hydroxylysine. In vitro, both peptides precipitate silica nanospheres within seconds when added to a monosilicic acid solution. Two silica-precipitating peptides, silaffin-1A1 and-1A2, both encoded by thesil1 gene from the diatom Cylindrotheca fusiformis, were extracted from cell walls and purified to homogeneity. The chemical structures were determined by protein chemical methods combined with mass spectrometry. Silaffin-1A1 and -1A2 consist of 15 and 18 amino acid residues, respectively. Each peptide contains a total of four lysine residues, which are all found to be post-translationally modified. In silaffin-1A2 the lysine residues are clustered in two pairs in which the ε-amino group of the first residue is linked to a linear polyamine consisting of 5 to 11N-methylated propylamine units, whereas the second lysine is converted to ε-N,N-dimethyllysine. Silaffin-1A1 contains only a single lysine pair exhibiting the same structural features. One of the two remaining lysine residues was identified as ε-N,N,N-trimethyl-δ-hydroxylysine, a lysine derivative containing a quaternary ammonium group. The fourth lysine residue again is linked to a long-chain polyamine. Silaffin-1A1 is the first peptide shown to contain ε-N,N,N-trimethyl-δ-hydroxylysine. In vitro, both peptides precipitate silica nanospheres within seconds when added to a monosilicic acid solution. high pressure liquid chromatography electrospray ionization mass spectrometry 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 (1Lowenstam H.A. Science. 1981; 211: 1126Crossref PubMed Scopus (983) Google Scholar). A large proportion of biogenic silica is formed by diatoms (2Tréguer P. Nelson D.M. van Bennekom A.J. DeMaster D.J. Leynaert A. Quéginer B. Science. 1995; 268: 375-379Crossref PubMed Scopus (1138) Google Scholar), which are unicellular algae that are ubiquitously present in marine and freshwater habitats (3Round F. Crawford R. Mann D. The Diatoms. Cambridge University Press, Cambridge1990Google Scholar). 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 (4Volcani B.E. Bendz G. Lindqvist I. Biochemistry of Silicon and Related Problems. Plenum Press, New York1978: 177-204Crossref Google Scholar, 5Robinson D.H. Sullivan C.W. Trends. Biochem. Sci. 1987; 12: 151-154Abstract Full Text PDF Scopus (56) Google Scholar, 6Kröger N. Sumper M. Bäuerlein E. Biomineralization. Wiley-VCH, Weinheim, Germany2000: 151-170Google Scholar). This has led to the discovery of silicic acid transporter proteins (7Hildebrand M. Volcani B.E. Gassmann W. Schroeder J.I. Nature. 1997; 385: 688-689Crossref PubMed Scopus (231) Google Scholar, 8Hildebrand M. Dahlin K. Volcani B.E. Mol. Gen. Genet. 1998; 260: 480-486Crossref PubMed Scopus (157) Google Scholar) and unique organic components that are associated with biosilica (9Kröger N. Lehmann G. Rachel R. Sumper M. Eur, J. Biochem. 1997; 239: 259-264Crossref Scopus (124) Google Scholar, 10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar, 11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar).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 (12Mann S. Ozin G. Nature. 1996; 382: 313-318Crossref Scopus (1131) Google Scholar, 13Parkinson J. Gordon R. Trends Biotechnol. 1999; 17: 190-196Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 14Cha J.N. Stucky G.D. Morse D.E. Deming T.J. Nature. 2000; 403: 289-292Crossref PubMed Scopus (637) Google Scholar). Recently, silica-associated components from different diatom species were identified that mediate the formation of silica nanospheres in vitro from a silicic acid solution (10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar, 11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). 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 (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). In contrast, there is only incomplete information about the chemical structure of the silaffins. Recently, a silaffin-encoding gene, termedsil1, 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 (10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar). 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 ε-amino group a linear polyamine consisting of 5 to 11 N-methylated propylamine units. Lysy has been shown to represent ε-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.DISCUSSIONThe 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 ε-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 ε-N,N,N-trimethyl-δ-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 (19Nakajima T. Volcani B.E. Biochem. Biophys. Res. Commun. 1970; 39: 28-33Crossref PubMed Scopus (31) Google Scholar) isolated and characterized for the first time ε-N,N,N-trimethyl-δ-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 ε-N,N,N-trimethyl-δ-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 (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar), whereas synthetic silaffin peptides lacking the lysine modifications are unable to precipitate silica at pH < 7 in vitro (10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar). In this respect it is interesting to note that silica formation in diatoms takes place in an acidic, intracellular compartment (20Vrieling E.G. Gieskes W.W.C. Beelen T.P.M. J. Phycol. 1999; 35: 548-559Crossref Scopus (162) Google Scholar), and thus the polyamine moieties of the silaffin-1A peptides appear to be essential to mediate silica precipitation under physiological conditions. The ε-N,N,N-trimethyl-δ-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 (21Burkett S.L. Davies M.E. Alberti G. Bein T. Comprehensive Supramolecular Chemistry. Pergamon, Oxford1996: 465-483Google Scholar). Possibly, the ε-N,N,N-trimethyl-δ-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 (22Mort A.J. Lamport D.T.A. Anal. Biochem. 1977; 82: 289-309Crossref PubMed Scopus (279) Google Scholar). Silaffins contain a large number of hydroxyamino acid residues, which may be targets forO-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-1A2sequences 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. 1 B). Remarkably, arginine is the biosynthetic precursor of putrescine (23Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar), and the latter has been shown to serve as the attachment site for long-chain polyamines in C. fusiformisand other diatoms (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). 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. 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 (1Lowenstam H.A. Science. 1981; 211: 1126Crossref PubMed Scopus (983) Google Scholar). A large proportion of biogenic silica is formed by diatoms (2Tréguer P. Nelson D.M. van Bennekom A.J. DeMaster D.J. Leynaert A. Quéginer B. Science. 1995; 268: 375-379Crossref PubMed Scopus (1138) Google Scholar), which are unicellular algae that are ubiquitously present in marine and freshwater habitats (3Round F. Crawford R. Mann D. The Diatoms. Cambridge University Press, Cambridge1990Google Scholar). 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 (4Volcani B.E. Bendz G. Lindqvist I. Biochemistry of Silicon and Related Problems. Plenum Press, New York1978: 177-204Crossref Google Scholar, 5Robinson D.H. Sullivan C.W. Trends. Biochem. Sci. 1987; 12: 151-154Abstract Full Text PDF Scopus (56) Google Scholar, 6Kröger N. Sumper M. Bäuerlein E. Biomineralization. Wiley-VCH, Weinheim, Germany2000: 151-170Google Scholar). This has led to the discovery of silicic acid transporter proteins (7Hildebrand M. Volcani B.E. Gassmann W. Schroeder J.I. Nature. 1997; 385: 688-689Crossref PubMed Scopus (231) Google Scholar, 8Hildebrand M. Dahlin K. Volcani B.E. Mol. Gen. Genet. 1998; 260: 480-486Crossref PubMed Scopus (157) Google Scholar) and unique organic components that are associated with biosilica (9Kröger N. Lehmann G. Rachel R. Sumper M. Eur, J. Biochem. 1997; 239: 259-264Crossref Scopus (124) Google Scholar, 10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar, 11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). 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 (12Mann S. Ozin G. Nature. 1996; 382: 313-318Crossref Scopus (1131) Google Scholar, 13Parkinson J. Gordon R. Trends Biotechnol. 1999; 17: 190-196Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 14Cha J.N. Stucky G.D. Morse D.E. Deming T.J. Nature. 2000; 403: 289-292Crossref PubMed Scopus (637) Google Scholar). Recently, silica-associated components from different diatom species were identified that mediate the formation of silica nanospheres in vitro from a silicic acid solution (10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar, 11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). 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 (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). In contrast, there is only incomplete information about the chemical structure of the silaffins. Recently, a silaffin-encoding gene, termedsil1, 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 (10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar). 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 ε-amino group a linear polyamine consisting of 5 to 11 N-methylated propylamine units. Lysy has been shown to represent ε-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. DISCUSSIONThe 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 ε-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 ε-N,N,N-trimethyl-δ-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 (19Nakajima T. Volcani B.E. Biochem. Biophys. Res. Commun. 1970; 39: 28-33Crossref PubMed Scopus (31) Google Scholar) isolated and characterized for the first time ε-N,N,N-trimethyl-δ-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 ε-N,N,N-trimethyl-δ-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 (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar), whereas synthetic silaffin peptides lacking the lysine modifications are unable to precipitate silica at pH < 7 in vitro (10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar). In this respect it is interesting to note that silica formation in diatoms takes place in an acidic, intracellular compartment (20Vrieling E.G. Gieskes W.W.C. Beelen T.P.M. J. Phycol. 1999; 35: 548-559Crossref Scopus (162) Google Scholar), and thus the polyamine moieties of the silaffin-1A peptides appear to be essential to mediate silica precipitation under physiological conditions. The ε-N,N,N-trimethyl-δ-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 (21Burkett S.L. Davies M.E. Alberti G. Bein T. Comprehensive Supramolecular Chemistry. Pergamon, Oxford1996: 465-483Google Scholar). Possibly, the ε-N,N,N-trimethyl-δ-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 (22Mort A.J. Lamport D.T.A. Anal. Biochem. 1977; 82: 289-309Crossref PubMed Scopus (279) Google Scholar). Silaffins contain a large number of hydroxyamino acid residues, which may be targets forO-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-1A2sequences 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. 1 B). Remarkably, arginine is the biosynthetic precursor of putrescine (23Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar), and the latter has been shown to serve as the attachment site for long-chain polyamines in C. fusiformisand other diatoms (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). 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. 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 ε-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 ε-N,N,N-trimethyl-δ-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 (19Nakajima T. Volcani B.E. Biochem. Biophys. Res. Commun. 1970; 39: 28-33Crossref PubMed Scopus (31) Google Scholar) isolated and characterized for the first time ε-N,N,N-trimethyl-δ-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 ε-N,N,N-trimethyl-δ-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 (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar), whereas synthetic silaffin peptides lacking the lysine modifications are unable to precipitate silica at pH < 7 in vitro (10Kröger N. Deutzmann R. Sumper M. Science. 1999; 286: 1129-1132Crossref PubMed Scopus (1203) Google Scholar). In this respect it is interesting to note that silica formation in diatoms takes place in an acidic, intracellular compartment (20Vrieling E.G. Gieskes W.W.C. Beelen T.P.M. J. Phycol. 1999; 35: 548-559Crossref Scopus (162) Google Scholar), and thus the polyamine moieties of the silaffin-1A peptides appear to be essential to mediate silica precipitation under physiological conditions. The ε-N,N,N-trimethyl-δ-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 (21Burkett S.L. Davies M.E. Alberti G. Bein T. Comprehensive Supramolecular Chemistry. Pergamon, Oxford1996: 465-483Google Scholar). Possibly, the ε-N,N,N-trimethyl-δ-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 (22Mort A.J. Lamport D.T.A. Anal. Biochem. 1977; 82: 289-309Crossref PubMed Scopus (279) Google Scholar). Silaffins contain a large number of hydroxyamino acid residues, which may be targets forO-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-1A2sequences 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. 1 B). Remarkably, arginine is the biosynthetic precursor of putrescine (23Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3221) Google Scholar), and the latter has been shown to serve as the attachment site for long-chain polyamines in C. fusiformisand other diatoms (11Kröger N. Deutzmann R. Bergsdorf C. Sumper M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14133-14138Crossref PubMed Scopus (587) Google Scholar). 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. We thank E. Hochmuth for expert technical assistance and LEO Elektronenmikroskopie (Oberkochen, Germany) for help with the scanning electron microscopy.