A Pattern Recognition Protein for Peptidoglycan

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect. The PGRP is an essential component for peptidoglycan to trigger the prophenoloxidase cascade that is now recognized to be an important insect defense mechanism. We cloned cDNA encoding PGRP from the silkworm fat body cDNA library. Northern blot analysis showed that the PGRP gene is constitutively expressed in the fat body, epithelial cell, and hemocytes of naive silkworms. Furthermore, a bacterial challenge intensified the gene expression, with the maximal period being from 6 to 36 h after infection. The upstream sequence of the cloned PGRP gene was shown to contain putativecis-regulatory elements similar to the NF-κB-like element, interferon-response half-element, and GATA motif element, which have been found in the promoters of the acute phase protein genes of mammals and insects. A homology search revealed that the homologs of silkworm PGRP are present in mice, nematodes, and bacteriophages. This suggests that the recognition of peptidoglycan as foreign is effected in both vertebrates and invertebrates by PGRP homologs with an evolutionally common origin. Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect. The PGRP is an essential component for peptidoglycan to trigger the prophenoloxidase cascade that is now recognized to be an important insect defense mechanism. We cloned cDNA encoding PGRP from the silkworm fat body cDNA library. Northern blot analysis showed that the PGRP gene is constitutively expressed in the fat body, epithelial cell, and hemocytes of naive silkworms. Furthermore, a bacterial challenge intensified the gene expression, with the maximal period being from 6 to 36 h after infection. The upstream sequence of the cloned PGRP gene was shown to contain putativecis-regulatory elements similar to the NF-κB-like element, interferon-response half-element, and GATA motif element, which have been found in the promoters of the acute phase protein genes of mammals and insects. A homology search revealed that the homologs of silkworm PGRP are present in mice, nematodes, and bacteriophages. This suggests that the recognition of peptidoglycan as foreign is effected in both vertebrates and invertebrates by PGRP homologs with an evolutionally common origin. lipopolysaccharide peptidoglycan β-1,3-glucan peptidoglycan recognition protein β-1,3-glucan recognition protein N-acetylmuramoyl-l-analine amidase (EC 3.5.1.28) base pair(s) kilobase pair(s) 1,4-piperazinediethanesulfonic acid Innate immunity plays vital roles in primary defense mechanisms against invading pathogens in both vertebrates and invertebrates. The common structures among pathogens are recognized to be non-selves in the immunity. This type of recognition is termed pattern recognition, as opposed to clonal recognition in which clonally selected immunoglobulins are employed (1Janeway Jr., C.A. Cold Spring Harbor Symp. Quant. Biol. 1989; 54: 1-13Crossref PubMed Google Scholar). Lipopolysaccharide (LPS)1 and peptidoglycan (PG) are the cell wall components of bacteria and β-1,3-glucan (βG) is that of fungi. They are recognized by pattern recognition proteins that are present in plasma as free-floating molecules or on the cell surface as receptors. Among the pattern recognition proteins in mammals, a humoral LPS-binding protein and the cellular receptor CD14 have been well characterized (2Schumann R.R. Leong S.R. Flaggs G.W. Gray P.W. Wright S.D. Mathison J.C. Tobias P.S. Ulevitch R.J. Science. 1990; 249: 1429-1431Crossref PubMed Scopus (1379) Google Scholar, 3Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3406) Google Scholar, 4Gegner J.A. Ulevitch R.J. Tobias P.S. J. Biol. Chem. 1995; 270: 5320-5325Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 5Viriyakosol S. Kirkland T.N. J. Biol. Chem. 1995; 270: 361-368Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Their roles in stimulating macrophages to produce cytokines are well understood. In contrast, pattern recognition proteins for PG and βG have not been characterized as thoroughly as those for LPS, although CD14 is also implicated for the recognition of PG as non-self (6Gupta D. Kirkland T.N. Viriyakosol S. Dziarski R. J. Biol. Chem. 1996; 271: 23310-23316Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 7Dziarski R. Tapping R.I. Tobias P.S. J. Biol. Chem. 1998; 273: 8680-8690Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). In insects, a number of proteins that could be pattern recognition proteins have been described. They are lectins (8Komano H. Natori S. Dev. Comp. Immunol. 1985; 9: 31-40Crossref PubMed Scopus (70) Google Scholar, 9Pendland J.C. Boucias D.G. Dev. Comp. Immunol. 1986; 10: 477-487Crossref PubMed Scopus (22) Google Scholar, 10Amanai K. Sakurai S. Ohtaki T. Comp. Biochem. Physiol. 1990; 97B: 471-476Google Scholar, 11Kotani E. Yamakawa M. Iwamoto S. Tashiro M. Mori H. Sumida M. Matsubara F. Taniai K. Okuda K.K. Kato Y. Mori H. Biochim. Biophys. Acta. 1995; 1260: 245-258Crossref PubMed Scopus (105) Google Scholar), hemolin (12Sun S.-C. Lindström I. Boman H.G. Faye I. Schmidt O. Science. 1990; 250: 1729-1732Crossref PubMed Scopus (215) Google Scholar,13Schmidt O. Faye I. Lindström-Dinnetz I. Sun S.-C. Dev. Comp. Immunol. 1993; 17: 195-200Crossref PubMed Scopus (36) Google Scholar), lipopolysaccharide-binding protein (14Jomori T. Natori S. J. Biol. Chem. 1991; 266: 13318-13323Abstract Full Text PDF PubMed Google Scholar, 15Koizumi N. Morozumi A. Imamura M. Tanaka E. Iwahana H. Sato R. Eur. J. Biochem. 1997; 248: 217-224Crossref PubMed Scopus (85) Google Scholar), Gram-negative bacteria-binding protein (16Lee W.-J. Lee J.D. Kravchenko V.V. Ulevitch R.J. Brey P.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7888-7893Crossref PubMed Scopus (191) Google Scholar), peptidoglycan recognition protein (PGRP) (17Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), and β-1,3-glucan recognition protein (βGRP) (18Ochiai M. Ashida M. J. Biol. Chem. 1988; 263: 12056-12062Abstract Full Text PDF PubMed Google Scholar, 19Söderhäll K. Rögner W. Söderhäll I. Newton R.P. Ratcliffe N.A. Insect Biochem. 1988; 18: 323-330Crossref Scopus (80) Google Scholar). The latter two proteins are members of the prophenoloxidase cascade, of which other members are known to be serine protease zymogens and prophenoloxidase (20Ashida M. Brey P.T. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman and Hall, London1997: 135-172Google Scholar). They have been shown to have specific affinity to PG or βG and to work at initiation points of the cascade. The prophenoloxidase cascade is now recognized to be one of major insect defense mechanisms and to possibly play vital roles in interrelating the mechanisms and in recognizing of microbes as foreign. Thus, it is probable that PGRP and βGRP of the prophenoloxidase cascade are employed generally in insects as pattern recognition molecules for PG and βG. We previously reported the purification and the characterization of silkworm PGRP and βGRP (17Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 18Ochiai M. Ashida M. J. Biol. Chem. 1988; 263: 12056-12062Abstract Full Text PDF PubMed Google Scholar). Pattern recognition proteins other than PGRP and βGRP are not known to be distributed among insects as widely as PGRP and βGRP. It is not clear whether pattern recognition proteins with the same specificity in mammals and in insects have any common structural similarity. However, parallels between cellular signaling pathways for the synthesis of mammalian acute phase proteins and insect immune proteins after a bacterial challenge have been revealed (21Hultmark D. Nature. 1994; 367: 116-117Crossref PubMed Scopus (67) Google Scholar). This indicates a common origin of the pathways in innate immunity in mammals and insects Here, we report the cloning of PGRP cDNA from the fat body cDNA library and PGRP gene from the genomic library of the silkwormBombyx mori. The homology search showed that PGRP is a homologous protein to bacteriophage T7 lysozyme, although it does not have lysozyme activity, and that proteins homologous to PGRP are expressed in mammals. This experimental evidence suggests that recognition proteins for the initial extracellular non-self recognition in innate immunity of vertebrates and invertebrates have developed from a common origin. Our experimental results also show that PGRP synthesis is induced by a bacterial challenge and suggest that expression of the PGRP gene is regulated by the Rel family of transcription factors. Silkworms, B. mori (strain Kinshu × Showa), were reared on an artificial diet as described previously (22Yoshida H. Ashida M. Insect Biochem. 1986; 16: 539-545Crossref Scopus (86) Google Scholar). The larvae on day 5 of the fifth instar were injected with 10 μl of late logarithmic phaseEnterobacter cloacae (JCM1232) suspension (A 600 = 0.1) in the physiological saline (10 mm bis-Tris propane buffer, pH 6.5, containing 150 mm NaCl) or with 10 μl of the saline as the control experiment. PGRP was purified as described by Yoshida et al. (17Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar).S-Pyridylethylated or intact PGRP was digested with trypsin at a molar ratio of enzyme to substrate of 1:50. The digestion was carried out in 0.1 m Tris-HCl, pH 6.5, at 37 °C for 24 h, and the resulting peptides were separated by high performance liquid chromatography on a C8 column (4.6 × 150 mm, Vydac). The isolated peptides were sequenced using an automatic protein sequencer (Shimadzu PSQQ-10). The sequences of the peptides derived from S-pyridylethylated or intact PGRP were compared to determine the disulfide bond locations. A silkworm fat body cDNA library was constructed in the vector λZAP (Stratagene). Using internal peptide sequences, degenerate oligonucleotides corresponding to KKQWDG and WPEWLE were synthesized. Their sequences were 5′-AAGAATTCAA(A/G)AA(A/G)CA(A/G)TGGGA(C/T)GG-3′ (sense primer) and 5′-AAGAATTCTC(A/C/G/T)A(A/G)CCA(C/T)TC(A/C/G/T)GGCCA-3′ (antisense primer), respectively. These primers were used for the polymerase chain reaction. The reaction was performed using the silkworm fat body cDNA library as a template under the following conditions: 35 cycles comprising 94 °C for 1 min, 60 °C for 2 min, and 72 °C for 3 min. A 480-bp fragment was amplified, subcloned into a plasmid vector pBluescript (Stratagene), and sequenced using an automatic DNA sequencer (PE Applied Biosystems, model 377). The cloned polymerase chain reaction product labeled with [α-32P]dCTP was used to probe the λZAP fat body cDNA library. Hybridization was carried out at 42 °C for 16 h in 2 × PIPES buffer (0.8 m NaCl, 40 mmPIPES, pH 6.5), 50% formamide, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. The membrane was washed twice in 0.1 × SSC (15 mm NaCl, 1.5 mm sodium citrate, pH 7.2) at 53 °C for 15 min and subjected to autoradiography. Three positive clones were obtained and sequenced. The clone that had the longest insert and a complete open reading frame was used as PGRP cDNA clone. In the detection of PGRP transcript in naive silkworms, poly(A)+ RNA preparations from hemocytes, fat body, epidermal cells, midgut, silk gland, and malpighian tubules were prepared by chromatography on oligo(dT)-cellulose (Amersham Pharmacia Biotech). 5 μg of poly(A)+ RNA of each preparation were separated by electrophoresis in 1% agarose gels with 10 mm sodium phosphate, pH 7.0, transferred to a Hybond-N+ (Amersham Pharmacia Biotech) membrane, and hybridized with a 32P-labeled PGRP cDNA probe. The hybridization was performed for 16 h at 42 °C in 50% formamide, 4 × SSPE (1.2 m NaCl, 40 mm sodium phosphate, pH 7.4, and 4 mm EDTA), 5 × Denhardt's (0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, and 0.1% Ficoll), 0.1% SDS, and 100 μg/ml denatured salmon sperm DNA. The membrane was washed once in 2 × SSPE containing 0.1% SDS and subsequently twice in 1 × SSPE containing 0.1% SDS at 56 °C for 10 min. In the experiments to demonstrate the induction of PGRP gene in the fat body, total RNA from the fat body of silkworms was collected at intervals after the bacterial challenge. 20-μg aliquots of the RNA preparations were subjected to Northern blot analyses as above except that silkworm cecropin B (0.4 kbp) (23Kato Y. Taniai K. Hirochika H. Yamakawa M. Insect Biochem. Mol. Biol. 1993; 23: 285-290Crossref PubMed Scopus (52) Google Scholar) and α-tubulin (1.3 kbp) probes were used in addition to the PGRP probe. The silkworm cecropin B (0.4 kbp) and α-tubulin (1.3 kbp) probes were obtained as polymerase chain reaction products, and the identities of their sequences to those having been reported were confirmed by referring to GenBankTM accession numbers S60579 and X83429, respectively. Silkworm genomic DNA was isolated from the silk gland using DNzol (Life Technologies, Inc.), and 10 μg of the DNA were digested with EcoRI, BamHI, orSacI. DNA fragments in the digests were separated by electrophoresis on a 0.8% agarose gel, blotted onto Hybond-N+ membrane, and hybridized with the32P-EcoRI/KpnI-digested cDNA. The digested cDNA probe (0.77 kbp) was the entire insert of PGRP cDNA clone with short flanking sequences. The conditions for the hybridization and the washings were the same as those in Northern blot analysis. 2 × 105plaques of an amplified B. mori genomic library constructed in λFIX (Stratagene) were screened with the 32P-labeled PGRP cDNA probe. Three positive plaques were isolated, and the λDNA was digested with BamHI or SacI. The32P-labeled PGRP cDNA probe hybridized with 2.5- and 4.3-kbp DNA fragments of BamHI digest and with a 2.1-kbp fragment of SacI digest. All the fragments were separately subcloned into pBluescript. After the deletion clones of the subclones were prepared, they were sequenced. Computer analysis of the sequencing data was performed using the GENETYX system (Software Development Co., LTD Tokyo). PGRP cDNA clones were obtained by screening a fat body cDNA library of the silkworm B. mori. The nucleotide sequence and the deduced amino acid sequence are shown in Fig. 1. The open reading frame composed of nucleotides amino acid The amino acid to be a peptide the deduced amino acid sequence from the was to that previously at the of Thus, the protein of with a of of the deduced protein the was confirmed to be to that by sequencing of peptides obtained after trypsin digestion of PGRP sequence for was not found in the deduced A was not of the The in the of the disulfide bond were to be and from a of the results of peptide and amino acid sequencing of and Northern blot analysis using PGRP cDNA as a probe a transcript of PGRP in hemocytes, fat body, and epidermal not in malpighian silk gland, and of naive silkworms. This indicates that PGRP is constitutively expressed in the fat body, epidermal cells, and for the synthesis of PGRP to be the fat body and epidermal The expression of PGRP gene and cecropin B gene in fat body was induced by of bacteria to silkworm as shown in B. The induction was similar in both the induction was at 6 h and at 24 h after saline was injected of bacteria in the above Northern of the of PGRP and cecropin B genes in the fat body for 36 h after the not These results that the expression of PGRP gene is by a bacterial challenge and that PGRP could be as an acute phase We could the induction of PGRP gene expression in the fat body by the We isolated positive clones from the silkworm genomic library. The of the clones that a 2.1-kbp SacI fragment both the 2.5- and 4.3-kbp BamHI fragments of fragments and the 2.1-kbp SacI fragment were subcloned separately into pBluescript. The sequences of fragments were after the of the nucleotide sequence of the PGRP gene with that of the PGRP cDNA the PGRP gene was revealed to contain nucleotides and by nucleotide of the PGRP cDNA clone in Fig. with The of is the of transcription of the PGRP gene not been The BamHI fragment of upstream sequence of the 3 The is from nucleotide to sequences were from the They were motif motif GATA motif and that have been found in of acute phase protein genes Y. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman and Hall, London1997: Scholar). blot analyses of genomic DNA digested with or SacI were performed by using the PGRP cDNA probe. is shown in Fig. 3 one hybridized was with the digest by and the digest with SacI two hybridized and the cloned PGRP gene had one for each of BamHI and the digest with BamHI have two hybridization in addition to the The we could not the 2.1-kbp fragment to be that the fragment a short and the PGRP cDNA probe hybridized with the Thus, the results of the blot analyses the of a of the PGRP gene in the silkworm We for the of silkworm PGRP homologs in the data base sequences by and found that an expressed sequence from the and sequences of bacteriophage T7 lysozyme and protein are homologous to silkworm PGRP Furthermore, the gene encoding RNA polymerase was found to contain a The sequence is in the of RNA polymerase sequence and to from the of the sequence We have previously reported the purification and characterization of the PGRP from the of the silkworm B. mori (17Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). The PGRP specifically binds to and to of the prophenoloxidase cascade in the plasma of the silkworm In the present we cloned PGRP cDNA and the The cloned PGRP cDNA an open reading frame encoding a protein with amino acid The deduced amino acid sequence of the protein had a acid sequence and a protein The from the primary of the deduced protein is in with the by electrophoresis of purified The sequence in the deduced amino acid sequence of PGRP is with that purified PGRP not to We have PGRP in a expression system and confirmed that the protein the to specifically to T. and M. This that cloned cDNA Among proteins of which the sequence and are bacteriophage T7 lysozyme is the protein with peptidoglycan and having the sequence with Furthermore, the amino acid of PGRP corresponding to the of the lysozyme Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar) were with other amino the of PGRP to to the homology is the same the present results the PGRP sequence not any to of does the of PGRP and peptidoglycan of PGRP to peptidoglycan is known to to the of protease zymogens of the prophenoloxidase cascade, we had that PGRP is a protease of which is by to However, the deduced PGRP sequence to We now that the of peptidoglycan and PGRP a protease that is a of the prophenoloxidase cascade. In the system of the that with as lipopolysaccharide and β-1,3-glucan are of T. S. B. in Molecular and Scholar). In the component at the initiation of the prophenoloxidase cascade is from that of the cascade. Our search for proteins with the sequence to PGRP that homologs to PGRP are present in and The protein been reported to be a J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). It to be whether the silkworm PGRP a similar The protein of the homologs to PGRP to have and in and to be present widely in This of PGRP suggests the that insect PGRP and homologs generally play a in the recognition of peptidoglycan in innate immunity. The of PGRP in the prophenoloxidase cascade be to insects. Peptidoglycan as have been in the of (17Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) and of by Dziarski and (6Gupta D. Kirkland T.N. Viriyakosol S. Dziarski R. J. Biol. Chem. 1996; 271: 23310-23316Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 7Dziarski R. Tapping R.I. Tobias P.S. J. Biol. Chem. 1998; 273: 8680-8690Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). the the for the recognition of peptidoglycan as foreign been understood. Dziarski and showed that CD14 on macrophages affinity to peptidoglycan and is in of the transcription by peptidoglycan R. Tapping R.I. Tobias P.S. J. Biol. Chem. 1998; 273: 8680-8690Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). It been that CD14 is a receptor for in mammals S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3406) Google Scholar). In the LPS-binding in plasma is to a with LPS, and of CD14 for the in with that for Thus, the LPS-binding protein the of with CD14 to It be whether PGRP in the same as the LPS-binding A of the PGRP gene was to be present in the silkworm 3 We a element, NF-κB-like element, and in the of the The of the gene were in the fifth instar larvae with Gram-negative bacteria or However, the transcription was after with the microbes or the cell wall component The elements an and the of transcription by cell wall components of microbes are common of acute phase proteins of insects and mammals M. M. E. J.A. J. 1993; PubMed Scopus Google Y. Sun Hultmark D. Faye I. J. Mol. Biol. 1993; PubMed Scopus Google Scholar). In PGRP be to be an acute phase immune expression of the PGRP gene bacterial challenge for silkworms those are to be The to the induction of acute phase immune protein synthesis are now being in both insects and mammals. to the Rel family are employed in the signaling for the induction in (21Hultmark D. Nature. 1994; 367: 116-117Crossref PubMed Scopus (67) Google Scholar, T. Immunol. 1994; PubMed Scopus Google U. K. Biol. 1994; 10: PubMed Scopus Google Scholar). been shown to be a receptor for a protein and to in the signaling for the immune protein gene for B. E. J.A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). a been reported to be in innate immunity as well as immunity R. Jr., C.A. Nature. 1997; PubMed Scopus Google Scholar). All to a common origin of the signaling Our present results the of homologous pattern recognition proteins for peptidoglycan in both insects and mammals. The results to that the for extracellular recognition of microbes as foreign is between insects and mammals, a between innate immune in as insects and mammals. the of the of the present a a cDNA for PGRP from a and and homologs was D. A. E. H. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar).