Properdin, the Positive Regulator of Complement, Is HighlyC-Mannosylated
2000; Elsevier BV; Volume: 275; Issue: 37 Linguagem: Inglês
10.1074/jbc.m001732200
ISSN1083-351X
AutoresSteffen Hartmann, Jan Hofsteenge,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoProperdin is the positive regulator of the alternative pathway of complement activation. The 53-kDa protein is essentially composed of six thrombospondin type 1 repeats, all of which contain the WXXW motif, the recognition sequence for C-mannosylation. C-Mannosylation is a post-translational modification of tryptophan residues in which, in contrast to the well known N- andO-glycosylation, the carbohydrate is attached via a C–C bond to C-2 of the indole moiety of tryptophan.C-Mannosylation was first found in human RNase 2 and interleukin-12. The terminal complement proteins C6–C9 also carry this modification as part of their thrombospondin type 1 repeats. We studied the C-mannosylation pattern of human properdin by mass spectrometry and Edman degradation. Properdin contains 20 tryptophans of which 17 are part of a WXXW motif. Fourteen tryptophans were found to be modified 100%. This is the first example of a protein in which the majority of tryptophan residues occurs in theC-mannosylated form. These results show thatC-mannosylated proteins occur at several steps along the complement activation cascade. Therefore, this system would be ideal to investigate the function of C-mannosylation. Properdin is the positive regulator of the alternative pathway of complement activation. The 53-kDa protein is essentially composed of six thrombospondin type 1 repeats, all of which contain the WXXW motif, the recognition sequence for C-mannosylation. C-Mannosylation is a post-translational modification of tryptophan residues in which, in contrast to the well known N- andO-glycosylation, the carbohydrate is attached via a C–C bond to C-2 of the indole moiety of tryptophan.C-Mannosylation was first found in human RNase 2 and interleukin-12. The terminal complement proteins C6–C9 also carry this modification as part of their thrombospondin type 1 repeats. We studied the C-mannosylation pattern of human properdin by mass spectrometry and Edman degradation. Properdin contains 20 tryptophans of which 17 are part of a WXXW motif. Fourteen tryptophans were found to be modified 100%. This is the first example of a protein in which the majority of tryptophan residues occurs in theC-mannosylated form. These results show thatC-mannosylated proteins occur at several steps along the complement activation cascade. Therefore, this system would be ideal to investigate the function of C-mannosylation. thrombospondin type 1 repeat high performance liquidchromatography interfaced withelectrospray ionizationmass spectrometry electrospray ionization tandem mass spectrometry phenylthiohydantoin aminoethylated cysteine Glycosylation is one of the most abundant and widespread post-translational modifications of proteins. In the common cases ofN- and O-glycosylation the glycan is attached via an amide or a hydroxyl group of an amino acid side chain to the protein. In human RNase 2 a fundamentally different type of glycosylation was discovered (1Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (247) Google Scholar, 2de Beer T. Vliegenthart J.F.G. Löffler A. Hofsteenge J. Biochemistry. 1995; 34: 11785-11789Crossref PubMed Scopus (99) Google Scholar, 3Löffler A. Doucey M.A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (67) Google Scholar). An α-mannosyl residue was found to be linked via a C–C bond to the C-2 of the indole ring of tryptophan (Fig. 1). It was shown that this modification is enzyme-catalyzed and that dolichylphosphate mannose is the sugar donor (4Doucey M.A. Hess D. Cacan R. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 291-300Crossref PubMed Scopus (142) Google Scholar). In RNase 2 the recognition signal for the transferase was determined to be WXXW (or less efficiently WXXF), in which the first tryptophan becomes modified (5Krieg J. Hartmann S. Vicentini A. Gläsner W. Hess D. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 301-309Crossref PubMed Scopus (122) Google Scholar). Meanwhile a total of 22 tryptophans in 7 proteins have been shown to be C-mannosylated, i.e. RNase 2 (1Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (247) Google Scholar), interleukin-12 (6Doucey M.A. Hess D. Blommers M.J. Hofsteenge J. Glycobiology. 1999; 9: 435-441Crossref PubMed Scopus (80) Google Scholar), and terminal complement proteins C6, C7, C8α and β, and C9 (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The terminal complement proteins showed a more complex pattern of C-mannosylated tryptophans than RNase 2 and interleukin-12. They contain as a part of their thrombospondin repeats (TSRs)1 WXXWXXW motifs, in which both of the first two tryptophans and, surprisingly, also the last one can be C-mannosylated. In addition, in TSRs of C6 and C7, tryptophans were found to be modified although they were not even part of a WXXW motif (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The complement system is an innate first line host defense mechanism against microbes. Its activation pathways are composed of three proteolytic cascades, which merge at the cleavage step of (inactive) C3, converting it into active C3b (Fig.2; reviewed in Ref. 8Prodinger W.M. Würzner R. Erdei A. Dierich M.P. Paul W.E. Fundamental Immunology. 4th Ed. Lippincott-Raven Publishers, Philadelphia1999: 967-995Google Scholar). Properdin (or factor P) is a positive regulator of the complement system. It stabilizes the C3 convertase (C3bBb) in the feedback loop of the alternative pathway (Fig. 2), protecting it from rapid inactivation (9Fearon D.T. Austen K.F. J. Exp. Med. 1975; 142: 856-863Crossref PubMed Scopus (317) Google Scholar, 10DiScipio R.G. Biochem. J. 1981; 199: 485-486Crossref PubMed Scopus (82) Google Scholar, 11Farries T.C. Lachmann P.J. Harrison R.A. Biochem. J. 1988; 252: 47-54Crossref PubMed Scopus (56) Google Scholar). In addition, the C5 convertase (C3bBbC3b), which converts C5 into C5b, can also bind properdin. This eventually leads to the formation of the membrane attack complex, the actual lytic moiety of complement that will kill the attacked microbe. The importance of properdin is demonstrated in properdin-deficient individuals. These patients have a higher susceptibility to meningococcal infections byNeisseria, leading to fulminant meningitis with mortality rates as high as 75% (reviewed in Ref. 12Linton S.M. Morgan B.P. Clin. Exp. Immunol. 1999; 118: 189-191Crossref PubMed Scopus (40) Google Scholar). Mature properdin monomer is a 53-kDa protein that occurs in plasma at a specific ratio of dimers, trimers, and tetramers of 26:54:20 (13Pangburn M.K. J. Immunol. 1989; 142: 202-207PubMed Google Scholar) and a concentration of 15–25 mg/liter. It consists of six TSRs, which are enclosed by N- and C-terminal parts (14Nolan K.F. Schwaeble W. Kaluz S. Dierich M.P. Reid K.B. Eur. J. Immunol. 1991; 21: 771-776Crossref PubMed Scopus (48) Google Scholar) that show no homology to other proteins. Five of the TSRs contain a WXXWXXWXXC motif, but in the fourth repeat the last tryptophan has been replaced by a valine. OneN-glycosylation site is found in the sixth TSR, which is not essential for activity of the protein (15Farries T.C. Atkinson J.P. J. Immunol. 1989; 142: 842-847PubMed Google Scholar, 16Higgins J.M. Wiedemann H. Timpl R. Reid K.B. J. Immunol. 1995; 155: 5777-5785PubMed Google Scholar). Finding C-mannosylation in the TSRs of the terminal complement proteins prompted the question of whether properdin, being composed mainly of TSRs, is also C-mannosylated. In this paper we show that properdin is the first protein in which the majority of tryptophans occurs in the C-mannosylated form. This, together with its clearly defined biological function, makes properdin an ideal protein for functional studies onC-mannosylation. Human properdin was purchased from Advanced Research Technologies (San Diego, CA).N-(Iodoethyl)trifluoroacetamide was from Sigma. Endoproteinase Lys-C (Achromobacter) was from Wako BioProducts (Richmond, VA). Properdin was reduced and aminoethylated according to Ref. 17Schwartz W.E. Smith P.K. Royer G.P. Anal. Biochem. 1980; 106: 43-48Crossref PubMed Scopus (38) Google Scholar. In brief, 50 μg of properdin were dissolved in 50 μl of 0.5 m Tris-HCl, 6 m guanidine HCl, 10 mm EDTA and reduced with 0.56 μmol of dithiothreitol for 4 h at room temperature under argon. After adding110 volume of methanol, the solution was heated at 50 °C. 3.1 μl of 4.5 m N-(iodoethyl)trifluoroacetamide dissolved in methanol were added twice and incubated at 50 °C for 60 min and 90 min, respectively. Removal of the trifluoroacetyl protection group was achieved by adding 40 μmol of acetic acid and incubating for 60 min at 37 °C. The protein was dialyzed against 50 mmTris-HCl, 1 m guanidine HCl, pH 9.0 containing 20% methanol, followed by the same buffer without methanol. Digestion with endoproteinase Lys-C was performed at 37 °C at an enzyme to substrate ratio of 1:40. After 20 h a fresh portion of protease was added, and the incubation was continued for another 20 h. Digests were fractionated by C8 reversed phase LC-ESIMS as described (18Krieg J. Gläsner W. Vicentini A. Doucey M.A. Löffler A. Hess D. Hofsteenge J. J. Biol. Chem. 1997; 272: 26687-26692Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Peptides containing an aminoethylated cysteine followed by a proline were not cleaved under these conditions. These peptides were further digested in the same buffer without guanidine HCl for 6 h at 37 °C with 500 ng of Lys-C. Fractionation by high pressure liquid chromatography at pH 6.0 was achieved as described (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Nano ESIMSMS and solid-phase Edman degradation were performed according to Refs. 19Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1719) Google Scholar and 20Pisano A. Redmond J.W. Williams K.L. Gooley A.A. Glycobiology. 1993; 3: 429-435Crossref PubMed Scopus (95) Google Scholar. The elution position of PTH-(C-2-Man-)Trp was established by comparison to standard runs of residues 5–10 of human RNase 2 [FT(C-2-Man-)WAQW] (1Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (247) Google Scholar) directly before or after analysis of each of the properdin peptides as described (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Peptides have been numbered as they occur in the sequence of the mature protein and labeled with the prefix "K" to indicate that they were generated by endoproteinase Lys-C. To obtain peptides from properdin that were suitable for analysis, we took advantage of the spacing of the cysteine residues of its TSRs by aminoethylation and endoproteinase Lys-C digestion. This yielded peptides ranging from 12 to 21 residues in length. Fractionation of an endoproteinase Lys-C digest of reduced and aminoethylated properdin was achieved by reversed phase LC-ESIMS (Fig.3). The mass spectrometry data were extracted for the masses of the expected peptides containing tryptophan(s) without and with one, two, or three hexosyl residue(s), which corresponds to an additional mass of 162 Da per hexosyl residue. The fractions were analyzed by ESIMSMS and Edman degradation. A peptide with a mass of 1928 Da was found to elute at 35.1 min, corresponding to peptide K52 (residues 353–364) with all three tryptophansC-mannosylated. In the same fraction a second peptide with a mass of 1149 Da was observed, which was identified by ESIMSMS as peptide K5 (GLLGGGVSVEDCae). ESIMSMS of the doubly charged ion of peptide K52 with an m/zvalue of 965 gave the spectrum shown in Fig.4. Three times the parent ion [M + 2H]2+ showed the loss of 120 Da (corresponding to anm/z value of 60 in the case of a doubly charged ion; indicated with dotted lines in Fig. 4). This loss of 120 Da is characteristic for aromatic C-glycosides (21Li Q.M. van den Heuvel H. Dillen L. Claeys M. Biol. Mass. Spectrom. 1992; 21: 213-221Crossref Scopus (78) Google Scholar, 22Becchi M. Fraisse D. Biomed. Environ. Mass Spectrom. 1989; 18: 122-130Crossref Scopus (92) Google Scholar) and has been instrumental in determining the presence of (C-2-Man-)Trp in several proteins (1Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (247) Google Scholar, 6Doucey M.A. Hess D. Blommers M.J. Hofsteenge J. Glycobiology. 1999; 9: 435-441Crossref PubMed Scopus (80) Google Scholar,7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Moreover, multiple water losses can be seen (labeled with #), which together with the 120-Da loss, form the fingerprint for aC-linked hexosyl residue. A nearly complete series of y ions together with several b ions confirmed the identity of peptide K52. For all y ions and for some b ions containing a C-mannosylated tryptophan, at least one loss of 120 Da (indicated with dashed lines), often together with the multiple water losses, was observed. This has been illustrated in Fig. 4. Edman degradation of this peptide confirmed that all tryptophans were C-mannosylated. Cycles 3, 6, and 9 of peptide K52 showed a PTH-derivative that comigrated with (C-2-Man-)Trp from RNase 2. No signal for the PTH-derivative of unmodified tryptophan was observed. The C-mannosylation pattern of this peptide is another example of the modification of the last tryptophan in a WXXWXXC context, as it was first found in the terminal complement proteins (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). These results have been summarized in Table I.Table ISummary of the evidence for C-mannosylated tryptophan in properdinPeptide1-aPeptides from an endoproteinase Lys-C digest have been numbered according to their occurrence in the polypeptide chain.Residue1-bResidue numbers are according to the mature protein.MassNumber of hexoses1-dNumber of expected hexoses: (observed mass − expected mass)/162 Da., 1-eSummary of spectra obtained from differently charged precursor ions. y and b ions that were observed are indicated with a line above and below the peptide sequence, respectively. Ions from which the 120-Da loss (which defines the presence of theC-mannose linkage) has been observed are indicated with a number that reflects the number of times a 120-Da loss occurred. W*, (C-2-Man-)Trp. Cysteines were aminoethylated.Position of modified TrpMSMS1-cExpected mass deduced from the cDNA sequence with aminoethylated cysteines.Retention time of C-mannosylated tryptophan PTH-derivativeExpected1-cExpected mass deduced from the cDNA sequence with aminoethylated cysteines.ObservedSampleRNase2DAView Large Image Figure ViewerDownload (PPT)1-a Peptides from an endoproteinase Lys-C digest have been numbered according to their occurrence in the polypeptide chain.1-b Residue numbers are according to the mature protein.1-c Expected mass deduced from the cDNA sequence with aminoethylated cysteines.1-d Number of expected hexoses: (observed mass − expected mass)/162 Da.1-e Summary of spectra obtained from differently charged precursor ions. y and b ions that were observed are indicated with a line above and below the peptide sequence, respectively. Ions from which the 120-Da loss (which defines the presence of theC-mannose linkage) has been observed are indicated with a number that reflects the number of times a 120-Da loss occurred. W*, (C-2-Man-)Trp. Cysteines were aminoethylated. Open table in a new tab At 40.3 min a peptide with a mass of 2099 Da was found that was 324 Da heavier than expected from the cDNA sequence for residues 49–66 (14Nolan K.F. Schwaeble W. Kaluz S. Dierich M.P. Reid K.B. Eur. J. Immunol. 1991; 21: 771-776Crossref PubMed Scopus (48) Google Scholar, 23Nolan K.F. Kaluz S. Higgins J.M. Goundis D. Reid K.B. Biochem. J. 1992; 287: 291-297Crossref PubMed Scopus (35) Google Scholar), corresponding to a modification with two hexosyl moieties. The pure peptide was directly analyzed by ESIMSMS and Edman degradation. The tandem mass spectrum of the doubly charged peptide twice showed a loss of 120 Da from the parent ion. Furthermore, a nearly complete series of b ions was observed, and all of the b ions containing a C-mannosylated tryptophan showed the 120-Da loss (Table I). Interestingly, the spectrum also showed a series of a ions, again with all possible 120-Da losses (data not shown). This experiment allowed us to localize the modified tryptophans to position 56 and 59 (Table I). The assignment was confirmed by Edman degradation. At 34.8 min a peptide with a mass of 2352 Da was found. This mass fitted to peptide K17–18 (residues 106–121), with an N-terminally aminoethylated cysteine and all three tryptophans C-mannosylated. The series of b and y ions observed in the ESIMSMS spectrum, the triple loss of 120 Da from the parent ion, and Edman degradation of the pure peptide confirmed this (Table I). A peptide with a mass of 2753 Da was found at 37.7 min that corresponded to the combination peptide K26–27 (residues 158–178) with two hexosyl residues. Apparently, endoproteinase Lys-C had not cleaved the (aminoethyl)Cys–Pro bond at position 163–164. ESIMSMS analysis showed two 120-Da losses from the parent ion. A series of b and y ions confirmed the identity of the peptide and localized the two modified tryptophans to positions 169 and 172 and one unmodified tryptophan to position 175. Edman degradation showed the same pattern of modified and unmodified tryptophans (TableI). In the case of peptide K37, endoproteinase Lys-C in the presence of guanidine HCl did not cleave the (aminoethyl)Cys–Pro bonds at positions 227–228 and 242–243. This resulted in the combination peptide K36–38 (eluting at 36.6 min), which was too large for analysis by ESIMSMS and Edman degradation. To obtain peptide K37, the peptide K36–38 was subjected to a Lys-C digest, but now without guanidine HCl. Its mass of 1834 Da matched that of peptide K37 (residues 228–242) modified with two hexosyl residues. The peptide was purified to apparent homogeneity by LC-ESIMS using a pH 6 buffer system (data not shown). The ESIMSMS spectrum showed two 120-Da losses from the parent ion and a complete y ion series with nearly all possible (C-2-Man-)Trp-related 120-Da losses. Edman degradation confirmed that both tryptophans areC-mannosylated (Table I). The sequence of interest was found in two different Lys-C cleavage products: the fully cleaved peptide K43 (at 36.9 min) and the combination peptide K42–43 (at 38.3 min). In both cases the masses were 324 Da heavier than predicted from the cDNA sequence, suggesting the presence of two hexosyl residues (Table I). The ESIMSMS spectra of both peptides showed two characteristic 120-Da losses from the parent ions. Both spectra allowed the assignment of the C-mannosylated tryptophans to position 294 and 297, with Trp-291 unmodified. The ESIMSMS spectrum of K43 showed a complete y ion series and a nearly complete b ion series, both with several secondary C-linked sugar losses. Edman degradation of K43 confirmed the modification pattern (Table I). The results presented in this paper (Fig.5) show that 14 of 20 tryptophans in properdin are stoichiometrically C-mannosylated. These tryptophans were identified by tandem mass spectrometry experiments and confirmed by Edman degradation (Table I). Mass spectrometry experiments cannot distinguish between different hexoses but enable the establishment of the presence of a C–C bond between the hexosyl residue and tryptophan by observing the 120-Da loss, typical for aromatic C-glycosides (21Li Q.M. van den Heuvel H. Dillen L. Claeys M. Biol. Mass. Spectrom. 1992; 21: 213-221Crossref Scopus (78) Google Scholar, 22Becchi M. Fraisse D. Biomed. Environ. Mass Spectrom. 1989; 18: 122-130Crossref Scopus (92) Google Scholar). Therefore, it was verified by Edman degradation that all PTH-derivatives of modified tryptophans comigrated with authentic PTH-(C-2-Man-)Trp (Table I). For three proteins, RNase 2 (2de Beer T. Vliegenthart J.F.G. Löffler A. Hofsteenge J. Biochemistry. 1995; 34: 11785-11789Crossref PubMed Scopus (99) Google Scholar, 3Löffler A. Doucey M.A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Biochemistry. 1996; 35: 12005-12014Crossref PubMed Scopus (67) Google Scholar), interleukin-12 (6Doucey M.A. Hess D. Blommers M.J. Hofsteenge J. Glycobiology. 1999; 9: 435-441Crossref PubMed Scopus (80) Google Scholar), and C9 (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), NMR always identified the hexosyl group to be α-mannose,C-glycosidically linked to the C-2 of the indole moiety. Importantly, the NMR data showed that in C9 this is true for both tryptophans in the WXXW motif (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Up to now noC-α-hexosylated tryptophan derivatives other than the mannosylated one have been detected; nor are synthetic compounds available. The recent synthesis of (C-2-Man-)Trp should also allow the production of other hexosyl derivatives, which would provide suitable controls (24Nishikawa T. Ishikawa M. Isobe M. Synlett. 1998; 1: 123-125Google Scholar, 25Manabe S. Ito Y. J. Am. Chem. Soc. 1999; 121: 9754-9755Crossref Scopus (72) Google Scholar). So far seven polypeptides have been shown to contain a total of 22C-mannosylated tryptophans. Properdin increases this number substantially, further supporting the notion thatC-mannosylation is not a rare post-translational modification (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In the case of RNase 2 (4Doucey M.A. Hess D. Cacan R. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 291-300Crossref PubMed Scopus (142) Google Scholar, 5Krieg J. Hartmann S. Vicentini A. Gläsner W. Hess D. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 301-309Crossref PubMed Scopus (122) Google Scholar) and interleukin-12 (6Doucey M.A. Hess D. Blommers M.J. Hofsteenge J. Glycobiology. 1999; 9: 435-441Crossref PubMed Scopus (80) Google Scholar) it has been well established that WXXW is the recognition signal for C-mannosylation, where only the first tryptophan becomes modified. In properdin, only the C-mannosylation of TSR 3 follows this rule (Fig. 5). In the other TSRs, tryptophans occur that are not followed by a second tryptophan (or another aromatic residue) at position +3. Most of the time a cysteine is found at this position; however, in TSR 4 a valine is present.C-Mannosylation of tryptophan residues in such a context has also been observed in the terminal components of complement. This led to the hypothesis that in addition to the WXXW motif, another signal must exist in these proteins (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). At present, it is not known what constitutes such a signal. In vitroexperiments using synthetic substrates strongly suggest that the additional signal is not formed by the residue at the +3 position (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). If this signal were solely encoded in the primary structure, the signal would lie outside the amino acid sequence used in these studies, and a second kind of C-mannosyltransferase would be involved (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Alternatively, the signal could be formed by a three-dimensional signal patch, as has been described for substrates of UDP-GlcNAc lysosomal enzyme:GlcNAc-1-phosphotransferase (26Baranski T.J. Koelsch G. Hartsuck J.A. Kornfeld S. J. Biol. Chem. 1991; 266: 23365-23372Abstract Full Text PDF PubMed Google Scholar). In TSRs 1 and 5 of properdin the first tryptophan is notC-mannosylated, indicating that negative signals must exist. These could be either neighboring amino acids or three-dimensional constraints. The high degree of C-mannosylation of properdin poses the question of degradation of (C-2-Man-)Trp. On the one hand, pathways could exist that degrade (C-2-Man-)Trp to intermediates suitable for entering metabolic pathways. On the other hand, (C-2-Man-)Trp could be excreted, either as part of a peptide or as free (C-2-Man-)Trp. The latter pathway is compatible with the recent finding of (C-2-Man-)Trp in human urine (27Gutsche B. Grun C. Scheutzow D. Herderich M. Biochem. J. 1999; 343: 11-19Crossref PubMed Scopus (64) Google Scholar). Considering the number ofC-mannosylated tryptophans in properdin and the terminal complement proteins (7Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and the concentrations of these proteins in plasma, the amounts of (C-2-Man-)Trp found in urine could readily be explained by turnover of only 5% of these proteins per day. 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In this way properdin is not only essential for the activation of the alternative pathway but also for the amplification of the classical and mannose-binding lectin pathways. Although there is no three-dimensional structure of properdin or a TSR module available, it seems very likely that the 14 C-linked mannoses, as hydrophilic moieties, will be exposed at the surface of properdin and therefore could influence the binding to C3b and Bb. Furthermore, it is known that many pathogenic microbes carry mannose-binding receptors on their surfaces (56Sharon N. Liener I.E. Sharon N. Goldstein I.J. The Lectins: Properties, Functions, and Applications in Biology and Medicine. Academic Press, Orlando, FL1986: 493-527Crossref Google Scholar). If properdin were to interact with these receptors through the C-linked mannoses and thereby get deposited on the surface of an invading microorganism, it could form a focus for attack through any of the three pathways of complement. Its stabilization of the C3 activation feedback loop would ensure local production of C3b, C5b, and the membrane attack complex. The same could be true for a possible interaction between the C-linked mannoses of properdin and the multivalent mannose-binding lectin from serum, which has been shown to bind a variety of microorganisms (57Turner M.W. Immunol. Today. 1996; 17: 532-540Abstract Full Text PDF PubMed Scopus (685) Google Scholar). Further studies are needed, however, as it is not known whether the (C-2-Man-)Trp residues in properdin are able to bind to lectins of bacterial or mammalian origin. In conclusion, it has been shown here that human properdin is a highlyC-mannosylated protein. The fact that, in addition to the terminal components C6-C9, the positive regulator properdin (with its well defined biological role) is C-mannosylated makes complement an ideal system to investigate the function of this post-translational modification. We thank Renate Matthies for amino acid sequencing, Dr. Daniel Hess for advice throughout the project, and Drs. Jack Rohrer and Daniel Hess for reading the manuscript. We also thank Dr. K. B. Reid (Oxford) for the initial gift of properdin.
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