Molecular characteristics of insect vitellogenins

ReviewMolecular characteristics of insect vitellogeninsMuhammad Tufail *,Makio TakedaGraduate School of Science and Technology,Kobe University,Nada,Kobe 657-8501,JapanContents 1.Vitellogenin—a food-source for the future embryo .....................................................................14472.Biochemical properties of insect vitellogenins .........................................................................14483.Vitellogenin genes in insects.......................................................................................14494.Protein architecture of insect Vgs...................................................................................14495.Phylogenetic analysis of insect vitellogenins ..........................................................................14526.Biosynthesis and post-transcriptional proteolytic cleavage of insect Vgs....................................................14537.Post-transcriptional glycosylation,phosphorylation,and sulphation .......................................................14548.Hormonal regulation of Vg gene transcription.........................................................................14559.Uptake of Vgs...................................................................................................145510.Conclusions ....................................................................................................1456References.....................................................................................................14561.Vitellogenin—a food-source for the future embryoAll oviparous species,including insects,provision their eggs with vitellin (Vn)as a major yolk protein.The precursorvitellogenin (Vg)is synthesized extra-ovarially by the fat body in tissue-,sex-,and stage-specific manners,secreted into the hemolymph and then sequestered by competent oocytes via receptor-mediated endocytosis (Raikhel and Dhadialla,1992;Sappington and Raikhel,1998;Snigirevskaya and Raikhel,2005).In the fat body,the Vg molecules are cleaved proteolytically and co-and post-translationally modified to facilitate the transport of carbohydrates,lipids,and other nutrients to the ovaries (seeJournal of Insect Physiology 54(2008)1447–1458A R T I C L E I N F O Article history:Received 9May 2008Received in revised form 14August 2008Accepted 14August 2008Keywords:Insect vitellogenins StructureCharacteristicsPost-transcriptional processing BiosynthesisTranscriptional regulation UptakeA B S T R A C TVitellogenins (Vgs)are precursors of the major egg storage protein,vitellin (Vn),in many oviparous animals.Insects Vgs are large molecules ($200-kD)synthesized in the fat body in a process that involves substantial structural modifications (e.g.,glycosylation,lipidation,phosphorylation,and proteolytic cleavage,etc.)of the nascent protein prior to its secretion and transport to the ovaries.However,the extent to which Vgs are processed in the fat body varies greatly among different insect groups.We provide evidence by cloning and peptide mapping of four Vg molecules from two cockroach species (Periplaneta americana and Leucophaea maderae )that,in hemimetabolous insects,the pro-Vg is cleaved into several polypeptides (ranging from 50-to 180-kD),unlike the holometabolans where the Vg precursor is cleaved into two polypeptides (one large and one small).An exception is the Vg of Apocrita (higher Hymenoptera)where the Vg gene product remains uncleaved.The yolk proteins (YPs)of higher Diptera (such as Drosophila )form a different family of proteins and are also not cleaved.So far,Vgs have been sequenced from 25insect species;9of them belong to Hemimetabola and 16to Holometabola.Alignment of the coding sequences revealed that some features,like the GL/ICG motif,cysteine residues,and a DGXR motif upstream of the GLI/CG motif,were highly conserved near thecarboxy terminal of all insect Vgs.Moreover,a consensus RXXR cleavage sequencemotif exists at the N-terminus of all sequences outside the Apocrita except for Lymantria dispar where it exists at the C-terminus.Phylogenetic analysis using 31Vg sequences from 25insect species reflects,in general,the current phylogenies of insects,suggesting that Vgs are still phylogenetically bound,although a divergence exists among them.ß2008Elsevier Ltd.All rights reserved.*Corresponding author.Tel.:+81788035869;fax:+81788035869.E-mail addresses:mtufail@kobe-u.ac.jp ,tufailm2001@ (M.Tufail).Contents lists available at ScienceDirectJournal of Insect Physiologyj o ur n a l h o m e p a g e :w w w.e l se v i e r.co m /l oc a t e /j i n s ph y s0022-1910/$–see front matter ß2008Elsevier Ltd.All rights reserved.doi:10.1016/j.jinsphys.2008.08.007reviews Raikhel and Dhadialla,1992;Hagedorn et al.,1998;Giorgi et al.,1999;Sappington et al.,2002;Tufail et al.,2005).After incorporation into oocytes,the Vgs are stored in a crystalline form as Vns,a reserve food-source for the future embryo.In insects,a female-specific protein was first found in the hemolymph of the silkmoth Hyalophora cecropia and was shown to participate in yolk formation by Tefler (1954).The major source of this protein was identified as the fat body and it was named ‘‘vitellogenin’’as the precursor of Vn or yolk protein (YP)by Pan et al.(1969).Later studies have shown that in insects the female fat body is the major site of Vg biosynthesis (Engelmann,1983;Wyatt and Davey,1996).Nevertheless,it has become clear that the fat body is not the only the vitellogenic tissue,as Cyclorapha ovarian follicular epithelium also produces YPs (Giorgi et al.,2005;Belles,1998,2005).In addition,Vg is no longer considered female-specific because its synthesis,although in smaller amounts,occurs in males of some species (Engelmann,1979;Trenczek and Engels,1986;Valle,1993;Piulachs et al.,2003).The Vgs have been studied extensively in a wide group of animals,both vertebrates and invertebrates including insects.For insects,Vgs have been sequenced from 25insect species belonging to six different orders (Table 1).Recently,we have cloned four Vg molecules from two cockroach species,Periplaneta americana and Leucophaea maderae (Tufail et al.,2000,2001,2005,2007;Tufail and Takeda,2002)and have shown by peptide mapping how these proteins undergo post-transcriptional proteolytic processing,and how quickly Vg subunits are sequestered by the oocyte when secreted into the hemolymph.In this review,we present the current understanding regarding Vg primary structure/character-istics,and mechanisms of its biosynthesis,transcriptional regula-tion,and post-transcriptional processing in insects.2.Biochemical properties of insect vitellogeninsInsect Vgs are phospholipoglycoproteins synthesized as $200-kD precursors derived from a 6–7-kb Vg mRNA (Table 2).The primary precursor undergoes proteolytic cleavage by dibasic endoproteases and splits into large (140–190-kD)and small (40–60-kD)subunits (see review Tufail et al.,2005).These subunits are assembled and secreted into the hemolymph as big oligomeric proteins (400–600-kD).Vgs generally exist as dimers,but monomeric molecules of about 300-kD are known from Nauphoeta cinerea (Imboden et al.,1987).However,in insects such as cockroaches,the Vg precursor is also cleaved into medium-sized ($90–110kD)polypeptides in addition to the large and small subunits (Tufail et al.,2001,2007;Tufail and Takeda,2002).Sometimes,Vgs are further processed in the ovary as reported in L.maderae (Tufail and Takeda,2002).Molecular studies have shown that Vgs from Hemimetabola are cleaved into several polypeptides unlike those from Holometabola where Vgs are either not cleaved as in Apocrita or cleaved only into two (one large and one small)polypeptides as in Aedes aegypti (see review Tufail et al.,2005).The YPs of higher Diptera are quite different from the Vgs of other insects and also are not processed.These are much smaller ($45-kD)and are related to the mammalian triglycerol lipase gene family (see Bownes and Pathirana,2002).The comparison of insect Vgs has shown that their structures are highly conserved (Chen et al.,1997;Sappington and Raikhel,Table 1Reference information of fully sequenced insect Vgs SpeciesOrderAccession numberNo.of genes identifiedAuthors(a)HemimetabolaPeriplaneta american -Vg1Dictyoptera AB034804Tufail et al.(2000)Periplaneta american -Vg2Dictyoptera AB0474012Tufail et al.(2001)Leucophaea maderae -Vg1Dictyoptera AB052640Tufail and Takeda (2002)Leucophaea maderae -Vg2Dictyoptera AB1949762Tufail et al.(2007)Blattella germanica Dictyoptera AJ0051151Comas et al.(2000)Lethocerus deyrollei Hemiptera AB4253341Nagaba,Tufail,Takeda (unpublished)Nilaparvata lugens Hemiptera AB3538561Naeemullah,Tufail,Takeda,Nakamura (unpublished)Riptortus clavatus Hemiptera U97272Hirai et al.(1998)Plautia stali -Vg1Hemiptera AB0334983Lee et al.(2000a)Plautia stali -Vg2Hemiptera AB033499Lee et al.(2000a)Plautia stali -Vg3Hemiptera AB033500Lee et al.(2000a)Graptopsaltria nigrofuscata Hemiptera AB0268481Lee et al.(2000b)Homalodisca coagulate HemipteraDQ1184081Hunter and Hunnicutt (unpublished)(b)HolometabolaAnthonomus grandis Coleoptera M729801Trewitt et al.(1992)Aedes aegyptiDiptera U025485Chen et al.(1994)and Romans et al.(1995)Anopheles gambiae Diptera AF2810782Romans (unpublished)Bombyx mori Lepidoptera D131601Yano et al.(1994a,b)Lymantria dispar Lepidoptera U907561Hiremath and Lehtoma (1997a,b)Antheraea pernyi Lepidoptera AB049631Liu et al.(2001)Antheraea yamamai Lepidoptera AB0558431Kajiura et al.(unpublished)Samia cynthiaLepidoptera AB0558441Kajiura et al.(unpublished)Bombyx mandarina Lepidoptera AB0558451Kajiura et al.(unpublished)Saturnia japonica Lepidoptera AB1908091Kajiura et al.(unpublished)Actias seleneLepidoptera EF5235671Liu et al.(unpublished)Athalia rosae Hymenoptera AB0078501Kageyama et al.(1994)and Nose et al.(1997)Pimpla nipponica Hymenoptera AF0267891Nose et al.(1997)Apis mellifera Hymenoptera AJ5174111Piulachs et al.(2003)Encarsia formosaHymenoptera AY5538781Donnell (2004)Solenopsis invicta -Vg1Hymenoptera AF5125203Lewis et al.(unpublished)Solenopsis invicta -Vg2Hymenoptera AY941795Tian et al.(unpublished)Solenopsis invicta-Vg3HymenopteraAY941796Tian et al.(unpublished)The accession numbers refer to database information linked to DDBJ.M.Tufail,M.Takeda /Journal of Insect Physiology 54(2008)1447–145814481998;Lee et al.,2000b)and form a gene superfamily.The similarities of Vgs/Vns among insects are also evident by their antigenicity.The antigenic similarity of Vns exists beyond the family level among21symphytan hymenopteran species(Taka-dera et al.,1996).However,in the pyralid moths,the antigenic similarity is limited to within the family level(Shirk,1987). Recently,the similarity in Vn-antigenicity among10cockroach species belonging to two superfamilies has been reported(Tufail et al.,2000;Tufail and Takeda,unpublished data).The similarity in Vn antigenicity is limited to within the superfamily in cockroaches except for that of L.maderae(Blaberoidea),where a90-kD Vn-polypeptide is antigenically related to that of P.americana (Blattoidea).Insect Vgs have also been shown to be homologous to Vgs of other organisms as divergent as nematodes and vertebrates(Blumenthal and Zucker-Aprison,1987;Trewitt et al.,1992;Chen et al.,1994).3.Vitellogenin genes in insectsInsect Vg genes are large and specify a single mRNA transcript of 6–7-kb(Table2).The number of Vg genes varies in different insect species(see review Tufail et al.,2005)(Table1).It has long been reported that L.maderae Vg is encoded by only a single Vg gene (Storella et al.,1985;Della-Cioppa and Engelmann,1987;Don-Wheeler and Engelmann,1991,1997),but we have recently cloned two Vg cDNAs from L.maderae and provided evidence that Vg in this cockroach is encoded by two different Vg genes(Tufail et al., 2007).Both genes(Vg1and Vg2)are transcribed since they could be amplified,through RACE-and RT-PCR,on mRNA from fat bodies of different individual females(Tufail et al.,2007).Both the L. maderae Vg gene primary products(pro-Vg1and pro-Vg2)share 96%similarity at the amino acid level.Two Vg molecules have also been characterized at the molecular and biochemical levels in P. americana(Tufail et al.,2000,2001,2005).Multiple Vg genes/ cDNAs have also been sequenced from other insect species including Locusta migratoria(Wyatt et al.,1984),Riptortus clavatus (Hirai et al.,1998),Plautia stali(Lee et al.,2000a),Solenopsis invicta (unpublished data),and Aedes aegypti(Romans et al.,1995)(for more detail see Tufail et al.,2005)(Table1).However,why some insects have multiple Vg genes is unclear.Different Vg genes may have different functions.For instance,the plasma clotting protein of crayfish is structurally homologous to Vgs but is responsible for clot formation in crustacean blood(Hall et al.,1999).Recently,it has been reported that Vg is involved in the regulation of hormonal dynamics and has multiple coordinating effects on the social organization of worker and male honey bees(Guidugli et al.,2005; Nelson et al.,2007).Information on the general and specific physiological roles of multiple Vg genes should prove to be of importance.4.Protein architecture of insect VgsSequence comparison of all insect Vgs has shown that they can be aligned confidently along their entire lengths.Subdomains I–V, areas of relatively high amino acid conservation(Chen et al.,1997; Sappington and Raikhel,1998;Lee et al.,2000b),were observed clearly(data not shown).The most striking characteristic of insect Vgs is the existence of polyserine tracts,which are at least conserved at the N-terminus of all sequenced Vgs(except Lymatria dispar and Anthonomus grandis)outside the apocritan Hymenop-tera(Fig.1).The cockroach and mosquito Vgs are still unique in harboring these tracts also at the C-terminus(Fig.1).However, their role is as yet unclear.The polyserine regions might serve as good phosphorylation sites.In P.americana and L.maderae,these domains were highly phosphorylated(Tufail et al.,2001,2005; Tufail and Takeda,2002).Phosphoserine tract represents extreme concentrations of negative charge(Goulas et al.,1996),which may promote the solubility of the Vg(Gerber-Huber et al.,1987)or chelation to essential metal ions such as Ca2+and Fe3+(Taborsky, 1991).Miller et al.(1982)and Dhadialla et al.(1992)reported that dephosphorylation of Vg reduces its uptake by oocytes,suggesting that phosphorylated residues may contribute to the interaction between Vg and its receptor on the oocyte surface.In all insects(except Apocrita),the primary Vg gene product is cleaved at a consensus cleavage site,R/K)XX(R/K,by subtilisin-like endoproteases(Barr,1991;Rouille et al.,1995).This tetra-residueTable2Identification of insect Vg gene transcriptsSpecies Method used Sex Stage Tissue Transcript size Authors(a)HemimetabolaDictyopteraP.american-Vg1NB Female Adult FB 6.5-kb Tufail et al.(2000)L.maderae-Vg1NB Female Adult FB 6.6-kb Tufail and Takeda(2002)B.germanica NB Female Adult FB 6.2-kb Martin et al.(1998)HemipteraR.clavatus NB Female Adult FB 6.3-kb Hirai et al.(1998)G.nigrofuscata NB Female Adult FB 6.8-kb Lee et al.(2000b)(b)HolometabolaColeopteraA.grandis NB Female Adult FB 6.0-kb Trewitt et al.(1992)DipteraAedes aegypti NB Female Adult FB 6.5-kb Chen et al.(1994)LepidopteraB.mori NB Female Larva/pupa FB 5.7-kb Yano et al.(1994a)L.dispar NB Female Larva FB 5.5-kb Hiremath and Lehtoma(1997a,b)HymenopteraA.rosae NB Female Adult FB 6.5-kb Kageyama et al.(1994)P.nipponica NB Female Adult FB 6.0-kb Nose et al.(1997)A.mellifera NB Female Pupa/adult FB 5.8-kb Piulachs et al.(2003)NB Male Adult FB 5.8-kb Piulachs et al.(2003)NB,Northern blot analysis;FB,fat body.M.Tufail,M.Takeda/Journal of Insect Physiology54(2008)1447–14581449motif is found conserved near the N-terminus of most of the non-Apocritan Vgs and is flanked by polyserine domains (Fig.1).However,in L.dispar ,the consensus RXXR sequence motif exists at the C-terminus.In hemimetabolous insects,where the Vg precursor is cleaved into several subunits,the RXXR motif was also determined near the C-terminus or at the center of the primary product (Hirai et al.,1998;Tufail et al.,2001,2005,2007;Tufail and Takeda,2002)(Fig.1).Comparison of the C-terminal part (subdomains IV and V)of all insect Vgs revealed a high degree of conservation of cysteine residues at 9positions following the GL/ICG motif (Fig.2).A similar but slightly longer motif (TCGLCG)was recognized in vertebrate Vgs (Mouchel et al.,1996),human von Willebrand factor (Baker,1988),and the human intestinal mucine 2glycoprotein (Gum et al.,1994).In addition,a DGXR motif is also located 17–19residues upstream of the GL/ICG motif in almost all (except L.maderae )insect Vg sequences (Fig.2).In N.lugens ,H.coagulata ,and S.invicta only arginine (R)is missing.The GL/ICG motif and cysteine residues are necessary for the oligomerization of vertebrate Vns (Mayadas and Wagner,1992;Mouchel et al.,1996).One possible function of oligomerized insect Vns could be binding lipids,in which inactive ecdysteroids are enclosed.The ecdysteroids are then released as Vns undergo proteolysis during embryogenesis (Hagedorn et al.,1998;Giorgi et al.,1999).It was proposed previously that the DG residues (of DGXR motif)together with GL/ICG motif and cysteine residues at conserved positions might form a structure necessary for Vns to function properly during embryogenesis (Tufail et al.,2001).Moreover,some amino acid residues like D and E (acidic),P (hydrophobic),and Y (hydrophilic)were also highly conserved at the C-terminus of almost all Vgs (Fig.2).However,their specific role at this position remainsunclear.Fig.1.Structural comparison of 27Vgs from 25insect species to match cleavage sites and polyserine stretches.Numbers indicate the amino acid residues deduced from the translation initiation methionine (excluding signal peptides).RXXR sequences are consensus cleavage sites (putative or determined)used during post-translational proteolytic processing.Nodes on the Vg-rods indicate the polyserine domains.For P.stali and S.invicta ,isoform 1has been shown (see Table 1for references).M.Tufail,M.Takeda /Journal of Insect Physiology 54(2008)1447–14581450M.Tufail,M.Takeda/Journal of Insect Physiology54(2008)1447–14581451Fig.2.Structural comparison of the C-terminal part of31Vgs.The alignment was performed using the Clustal W program and adjusted by eye.Dashes represent spaces introduced for maximum alignment.Numbers indicate the amino acid positions from the translation initiation methionines excluding the signal peptides.The DGXR and GL/ ICG motifs and cysteine-residues are shown with dark-shaded frames.The consensus sequence indicates residues common in most of the Vg sequences.The arrangement of insect names is the same given as in Fig.1.5.Phylogenetic analysis of insect vitellogeninsThe evolutionary relationship of 31Vgs derived from 25insect species was evaluated after aligning the complete amino acid sequences (without signal peptide)and conducting a phylogenetic analysis using neighbour-joining methods with MEGA version 3.1(Fig.3).The phylogenetic tree separated hemimetabolous from holometabolous Vg sequences with confidence into separate clusters (Fig.3A).Moreover,the tree clusters different insect species into specific orders in a quite coherent manner.Only cicada,G.nigrofuscata forms a cluster,although with only an 80%bootstrap value,with cockroaches as also reported recently by Tufail et al.(2007).We also tried the same analysis using subdomains I–V (after removing the variable regions)following Chen et al.(1997)and Sappington and Raikhel (1998)(Fig.3B).In this analysis,the Hemiptera clusters with the as et al.(2000)reported that the orders were incongruently distributed when the phylogenetic tree was constructed based on the subdomains of Chen et al.(1997)and suggested the use of the entire Vg sequence while conducting phylogenetic analyses.TheFig.2.(Continued ).M.Tufail,M.Takeda /Journal of Insect Physiology 54(2008)1447–14581452phylogenetic analysis based on a few or all five subdomains might be good for only one group of insects,as analyzed by Chen et al.(1997)and Sappington and Raikhel (1998).They used Vg sequences limited to Holometabola in their phylogenetic analyses.Several authors have used the entire Vg sequences to investigate the phylogenic relationship of Vgs among various insect species.Previously,Nose et al.(1997)constructed a phylogenetic tree using the complete amino acid sequences of Vgs from 6insect species.The tree was in agreement with the accepted phylogenies based on morphological characteristics and ribosomal DNA sequences.Subsequently,Lee et al.(2000b)constructed a phylogenetic tree based on the complete amino acid sequences of 9insects (3hemimetabolous and 6holometabolous);the results were similar to those reported by Nose et al.(1997).Tufail and Takeda (2002)reported a phylogenetic (neighbour-joining)tree based on the entire sequences of 4Vgs from three cockroach species (P.americana ,L.maderae and B.germanica ).The tree was in agreement with another molecular tree constructed based on the DNA sequences of mitochondrial 12S rRNA genes (Tufail and Takeda,2002)and with the most widely accepted phylogeny of cock-roaches constructed by Mckittrick (1964).A recent phylogenetic analysis using 23complete Vg sequences from 19insect species by Tufail et al.(2007)and the present phylogenetic tree (Fig.3A)reflect,in general,the current phylogenetic classification of insects,suggesting that Vgs are still phylogenetically bound although there exists a divergence among them.The results of the present phylogenetic analysis along with those reported by Comas et al.(2000)also suggest that the entire Vg sequences should be used when searching for phylogenetic inferences.6.Biosynthesis and post-transcriptional proteolytic cleavage of insect VgsVg is synthesized along the ribosomes associated with the rough endoplasmic reticulum,subsequently transferred to the Golgi apparatus,and eventually packaged into secretory granules emerging from the trans-Golgi network (see Mazzini et al.,1989;Snigirevskaya et al.,1997;Giorgi et al.,2005).During this process,the nascent Vg molecule is post-translationally modified by proteolytic cleavage (Sappington and Raikhel,1998;Tufail et al.,2005).Studies on insect Vgs show that they are synthesized as one or more large precursors ($200-kD),which are cleaved into two or more (depending on the species)subunits of smaller size,as discussed above.These subunits are assembled and secreted together as high molecular weight oligomeric proteins (Della-Cioppa and Engelmann,1987).In hemimetabolous insects,the pro-Vg is cleaved into several polypeptides ranging from 50to 180-kD,unlike the holometabo-lans where the Vg precursor is cleaved into two polypeptides (one large and one small).For example,in P.americana ,two Vg precursors are cleaved soon after their synthesis into three major [170-,100-(multisubunits),and a 50-kD]and a minor (150-kD)polypeptides (Tufail et al.,2000,2001,2005).The two Vg genes start being transcribed in 2-day-old adult-female fat body cells,whereas Vg is first detected in the hemolymph by immunoblotting in 4-day-old adult females,2days after the transcript first appeared (Tufail et al.,2000;Elgendy et al.,unpublished data),suggesting that the synthesis of Vg is regulated at the transcrip-tional level in this species.Moreover,in L.maderae ,five Vg polypeptides are produced (112-,100-,92-,87-,and 50-kD),which are encoded by two Vg genes (Vg1and Vg2)(Tufail et al.,2007).These cleavage products are assembled and secreted as a dimer (Della-Cioppa and Engelmann,1987).The post-transcriptional processing pattern of L.maderae Vg precursor molecules is summarized in Fig.4.In holometabolous insects,such as Aedes aegypti ,the native Vg has a molecular weight of 337-kD,which is comprised of two polypeptides of 200-and 66-kD (Dhadialla and Raikhel,1990).Bose and Raikhel (1988)have shown using an in vitro translation of the vitellogenic fat body mRNA that both Vg polypeptides are derived from a common precursor of 220-kD and thus originate from the samegene.Fig.3.Neighbour-joining phylogenetic analysis of 31Vg sequences.A distance analysis of amino acid sequences was performed using the Clustal W program and used as the input for a neighbour-joining tree construction program (MEGA3.1).Scale indicates distance (number of amino acid substitutions per site).(A)Phylogenetic tree constructed based on the entire Vg sequences (without signal peptide).(B)Phylogenetic tree constructed based on subdomains I–V (see Chen et al.,1997;Sappington and Raikhel,1998).Subdomains I–V were combined,after removing the variable regions,and then used for a single phylogenetic run.Numbers in the nodes correspond to bootstrap values in 100replicates.Source of amino acid sequences is given in Table 1.M.Tufail,M.Takeda /Journal of Insect Physiology 54(2008)1447–145814537.Post-transcriptional glycosylation,phosphorylation,and sulphationIn addition to proteolytic cleavage,the Vg molecules are co-and post-translationally modified to facilitate the transport of carbohydrates,lipids,sulphates,and other nutrients in the hemolymph to the ovaries (see reviews Raikhel and Dhadialla,1992;Hagedorn et al.,1998;Giorgi et al.,1999;Tufail et al.,2005).These conjugates not only support the embryo but also are involved somehow in internalization of the carrier into the oocyte.All insect Vgs characterized so far are glycosylated (see Giorgi et al.,1998;Tufail et al.,2005).In L.maderae ,all mature Vg polypeptides are glycosylated (Don-Wheeler and Engelmann,1997),and the attached carbohydrates are exclusively N-linked mannose oligosaccharides (Konig et al.,1988),which was confirmed by the existence of 14putative N-linked glycosylation sites (NXS/T)in both L.maderae Vgs (Tufail and Takeda,2002;Tufail et al.,2007).Such putative glycosylation sites were found also in both Vg molecules of P.americana (Tufail et al.,2000,2001),which included 20and 17sites,respectively.Studying the role of glycosylation in Vg synthesis,processing,and secretion from L.maderae ,Don-Wheeler and Engelmann (1997)demon-strated that the blocking of glycosylation with tunicamycin produced an aglycosylated Vg precursor of 190kD,which was accumulated within the fat body,as reported also from Locusta ,Aedes ,and Blattella (Wyatt et al.,1984;Wojchowski et al.,1986;Dhadialla and Raikhel,1990),suggesting that glycosylation is an important step in the subsequent secretion of Vg by the fat body.Moreover,Konig et al.(1988)suggested a complex role for covalently bound carbohydrate.In that,glycopeptide prepared by protease digestion of Vg inhibited binding of the intact protein,but after a -mannosidase treatment,whichremovesFig.4.Biosynthesis and post-transcriptional processing pathway of L.maderae Vgs (Pro-Vg1and Pro-Vg2).Both Vg precursors are synthesized and then cleaved into five subunit polypeptides (55-,100-,92-,112-and 87-kD)in the fat body before being secreted into the hemolymph (see Tufail et al.,2007).The 92-kD Vn polypeptide in the oocyte is further processed and results in the production of an additional 90-kD Vn polypeptide.The origin and position of all Vg/Vn subunits compared to their products is shown in (B).Post-translational cleavage sites (complete,C or partial,P)following RXXR consensus cleavage sequences (shown with arrows)for both L.maderae Pro-Vgs are indicated with dotted lines.Possible but unknown cleavage sites at the C-termini of 92-and 112-kD polypeptides (to generate the 90-and 87-kD subunits,respectively)are marked with question marks (see Tufail et al.,2007).Nodes on the Vg-rods indicate the polyserine tracts.SP is the signal peptide.Numbers on the right indicate the amino acids deduced from the translation initiation methionine (reproduced from Tufail and Takeda,2002;Tufail et al.,2007).M.Tufail,M.Takeda /Journal of Insect Physiology 54(2008)1447–14581454。