Patent Application: US-37633099-A

Abstract:
the present invention relates to a method for determining the effect of a test sample on uggt activity . the method comprises the steps of : a ) exposing an acceptor substrate of uggt such as acid phosphatase to a labeled donor such as udp - 3 h - glucose in the presence of the test sample and uggt ; and b ) detecting the amount of donor which was transferred to the acceptor substrate wherein a decrease of donor intake when compared to a control means that the test sample is a uggt stimulator and a decrease means that the test sample is a uggt inhibitor . the present invention also relates to an isolated mammalian cdna which encodes for rat uggt and to methods of producing mammalian uggt using recombinant vectors .

Description:
prior to setting forth this invention it may be helpful to first define certain terms that will be used herein . “ test sample ” means a sample to be assayed in the method of the present invention . the sample can include one or more compounds and can be a dry solid or a solution . the test sample can also be formulated when necessary . “ a uggt acceptor substrate ” means an unfolded protein which is found in the [ glcnac ] 2 ( man ) 7 - 9 form and which can be glucosylated by uggt by methods well known in the art . for example the substrate can be labeled by glycoprotein [ glcnac ] 2 ( man ) 7 - 9 denatred + udp -[ 3 h ] glucose + uggt . “ labeled donor ” means a nucleoside diphosphate which is a suitable donor substrate for uggt . “ folded protein ” means a secretory protein in its biologically active form . “ unfolded protein ” means a secretory protein in a non biologically active form or no longer in a bioactive form . in a preferred embodiment of the present invention the uggt acceptor substrate is selected from a group consisting of : yeast acid phosphatase , bovine pancreatic rnase b , bovine thyroglobulin , soybean agglutinin , yeast exo ( b - 1 , 3 ) glucanase and a yeast α - galactosidase . more preferably the uggt acceptor substrate is yeast exo ( b - 1 , 3 ) glucanase , α - galactosidase and rnase b . in a preferred embodiment of the present invention the labeled donor is udp - 3 h - glucose . the general conditions necessary for the method of the present invention are as follows : in accordance with the present invention there is provided an isolated nucleic acid comprising a cdna as set forth in seq id no : 1 . in accordance with the present invention there is provided a recombinant vector comprising the isolated nucleic acid of the present invention . in accordance with the present invention there is provided a host cell transfected with a recombinant vector comprising the isolated nucleic acid of the present invention . in accordance with the present invention , there is provided a process for preparing a recombinant mammalian uggt comprising the steps of : a ) culturing a host cell transfected with a recombinant vector comprising the isolated nucleic acid of the present invention under conditions wherein the said nucleic acid is expressed ; and preferably , the conditions are elution profile of rugt from ni ++− nta superflow column . the insect cell culture medium was concentrated , buffer exchanged and loaded on the column in buffer containing 10 mm imidazole . proteins were eluted using 20 mm imidazole in 40 mm tris - hcl ph 7 . 5 , 500 mm nacl , 5 mm cacl 2 . ( b ) elution profile of rugt from poros 20hq column . rugt fractions from the ni ++− nta column were pooled , diluted 10 fold and loaded on poros 20hq . rugt was eluted using an nacl gradient of 0 - 600 mm over 30 column volumes . the arrowhead indicates the rugt peak . preferably the host cell is a mammalian cell or an insect cell . more preferably the host cell is sf9 insect cells . the eukaryotic host cells appropriate for use in the invention include , preferably , vertebrate cells and , more preferably , mammalian cells . murine cell lines are especially preferred . however , in addition , other eukaryotes such as yeast cells may also be employed . the elements of the recombinant vector are constructed using standard recombinant dna techniques . the recombinant vectors of the present invention comprise a transcriptional promoter which is upstream of and operably linked to the isolated nucleic acid of the present invention . by “ operably linked ” is meant that the elements are ligated in such a fashion that their intended functions may be fulfilled . thus , the promoter “ operably linked ” to the isolated nucleic acid of the present invention is ligated in such a position and manner as to be capable of effecting the transcription of these dnas into mrna . typically a host expression vector is constructed which includes the transcription promoter operably linked to the isolated nucleic acid of the present invention followed by termination control sequences such as transcription terminator sequences and polyadenylation sites . these termination controls can be supplied from appropriate host sources such as those that control the termination of transcription of eucaryotic mrnas such as sv40 mrnas . typical polylinker sequences for gene insertion can be constructed synthetically and will include a variety of restriction sites . the expression vectors constructed according to the method of the invention are transfected or transformed into suitable recombinant host cells which are then cultured under conditions which permit the regulated production of the mammalian uggt . the choice of host will depend on the nature of the transcription and translation - regulating elements selected for the expression system . typically , the transfected cells are cultured under conditions where expression is repressed until a high density of cells is achieved . then conditions appropriate for the induction of expression are superimposed on the culture and protein production is commenced . the mammalian uggt produced is then recovered either from the supernatant or by cell lysis and purified using conventional means . in accordance with the present invention , there is provided a cdna or an analog thereof wherein said cdna or analog thereof encodes for uggt . in accordance with the present invention , there is provided the first cloned mammalian uggt successfully expressed in a baculovirus / insect cell system . active rugt was purified to homogeneity and exhibits the same preference for unfolded glycoproteins charged with n - linked [ glcnac ] 2 -( man ) 9 glycans as the enzyme purified from rat liver . the enzyme shows homology to other uggts throughout the protein but more so in the c - terminal 300 residues of the d . melanogaster , c . elegans and s . pombe gene products . the rat liver uggt is the first mammalian uggt to be sequenced . it shows a high degree of homology with the three other available sequences from the yeast schizosaccharomyces pombe , the nematode ceanorabditis elegans , and the fruit fly drosophila melanogaster , with 31 %, 43 % and 43 % of identity respectively , reflecting the evolutionary distance between these organisms ( fig9 a ). the sequence homology , although extending over the entire protein , is particularly high in a c - terminal region of about 300 residues from the end of the protein where the identity between the rugt sequence and that of the s . pombe uggt is 50 %. a domain homologous to this c - terminal segment was found in other glycosyltransferases from various organisms such as bacterial lipopolysaccharide glucosyl - and galactosyltransferases and mammalian glycogenins . a few other proteins of unknown functions also appear to contain a similar domain . the uggts , the bacterial lipopolysaccharide , monosaccharide transferases and the glycogenins all catalyze the transfer of a monosaccharide from a nucleoside diphosphate donor onto a variety of different substrates . it is therefore likely that their regions of homology would contain the active site where the donor substrate is bound , while the rest of the protein may be involved in the recognition of the acceptor substrate . two short stretches are particularly conserved , corresponding to residues 1329 to 1351 , and 1428 to 1435 of the mature rugt ( fig9 b ). four residues are entirely conserved suggesting a potential role in the function of these various proteins . in rugt , these residues are d1334 , d1336 , q1429 , and n1433 . to test if these residues are important for the function of rugt , they were all individually mutated for alanines ( fig8 ) and the activity of the resulting proteins was determined . these four amino acid residues appear to be involved at various degrees in catalytic activity . the first two residues completely abolish enzyme activity when they are mutated to alanines . the other two mutants retain about 2 % and 20 % of the activity of the wild - type . these residues are responsible either for the binding of the donor - substrate udp - glucose to rugt or in the actual catalytic transfer of the glucose residue to the acceptor - substrate . two models emerge whereby rugt would glucosylate every n - linked glycan on the acceptor - substrate like beads on a string or it may only recognize and glucosylate one or very few key sites along the denatured polypeptide chain . the first model could explain the possible substrate / product inhibition observed in the experiment designed to determine the km for acid phosphatase as incompletely glucosylated substrate molecules would remain bound to the enzyme thus preventing binding of other substrate molecules . the second model is much more appealing in that it implies that the oligosaccharide alone cannot constitute a complete target for rugt and that other peptide - specific determinants exposed in the unfolded polypeptide chain have to be recognized as well . this selective glucosylation model would imply the existence of specific / strategic sites for the binding of calnexin and / or calreticulin in the mechanism of refolding of glycoproteins in the er . in accordance with the present invention there is provided a strain of s . cerevisiae capable of producing asparagine - linked [ glcnac ] 2 -( man ) 9 glycoproteins . acid phosphatase , a naturally secreted yeast protein , was overexpressed in this strain and constitutes an excellent source of substrate for uggt . for expression in baculovirus / insect cells , the full cdna encoding rugt was subcloned in pfastbac - 1 . the native signal sequence of rugt was substituted for the honeybee melittin signal peptide to improve secretion ( tessier 1991 ). furthermore , the er retention signal heel at the c - terminal end of the protein was replaced by 6 histidine residues to allow release from the er and facilitate purification . at 72 hours post - infection , approximately 50 % of the expressed rugt was secreted in the insect cell culture medium ( fig8 ). the secreted form of rugt was purified to approximately 40 % homogeneity by immobilized metal - chelate affinity chromatography and further to greater than 95 % by anion exchange chromatography ( fig3 ; lane 6 ). from 400 ml of culture medium , 500 μg of rugt could be purified to near homogeneity as judged by coomassie - blue staining ( fig3 ). similar yields were obtained with all four rugt mutants d1334a , d1336a , q1429a , and n1433a indicating that the mutations do not appear to affect the stability of the enzyme . to produce a substrate for rugt , the s . cerevisiae strain yns - 7a known to produce mainly [ glcnac ] 2 -( man ) 8 oligosaccharides was further deleted of its mns1 gene which encodes an er α - mannosidase responsible for the trimming of the [ glcnac ] 2 -( man ) 9 oligosaccharides to [ glcnac ] 2 -( man ) 8 ( jelinek - kelly , 1988 and camirand , 1991 ). the resulting strain was called dt111 . secreted proteins from yns - 7a and dt111 were found to be much better substrates for rugt than proteins secreted from the wild - type w303 - 1a strain regardless of the growth conditions ( fig4 ). furthermore , the incorporation of 3 h - glucose was even better in proteins secreted from dt111 than from yns - 7a , as [ glcnac ] 2 -( man ) 9 glycans are known to be better acceptors than [ glcnac ] 2 -( man ) 8 ( sousa , 1992 ). these glycosylation deficient strains could eventually be used to produce recombinant proteins with tailored oligosaccharides . the yeast protein , acid phosphatase was a good candidate as it is highly glycosylated . moreover , acid phosphatase loses activity at neutral ph suggesting that it may unfold under these conditions , thus by - passing the need for a denaturation step prior to the glucosylation reaction . four acid phosphatase isoforms are encoded by four different genes pho3 , pho5 , pho11 and pho12 ( bajwa , 1984 ; arima , 1983 ; bussey , 1995 ; johnson , 1994 ). pho5 is responsible for the majority of the acid phosphatase synthesized and is further derepressed when cells are starved for inorganic phosphate ( tait - kamradt , 1986 ). the pho5 gene product has a predicted molecular weight of 51 . 1 kda , a theoretical pi of 4 . 60 and has 12 potential glycosylation sites . under inorganic phosphate starvation , the secretion of high molecular weight proteins seems to be stimulated in strain w303 - 1a ( fig4 ). these proteins appearing as a smear on sds - page are expected for acid phosphatase because it would be heterogenously hyperglycosylated in a wild - type strain . in yns - 7a and dt111 , the secretion of a protein of about 80 kda was increased under inorganic phosphate starvation . this molecular weight would be expected for acid phosphatase having [ glcnac ] 2 -( man ) 8 and [ glcnac ] 2 -( man ) 9 core oligosaccharides respectively . although crude culture supernatants from dt111 could be used as substrate for rugt , a simple change of buffer by ultrafiltration followed by anion exchange chromatography could yield a single protein of ˜ 80 kda . n - terminal sequencing of the purified protein has revealed the identity of acid phosphatase . complete treatment of purified acid phosphatase with endoh resulted in a reduction of & gt ; 20 kda of the apparent molecular weight on sds - page indicative of ˜ 12 glycosylation sites ( data not shown ). about 7 mg of & gt ; 90 % pure acid phosphatase could be obtained from 14 liters of dt111 cells grown in sd — pi medium in a bioreactor to a maximum o . d . 600 nm of 2 . the activity of the purified recombinant rugt was tested towards various substrates . as expected the enzyme showed a greater activity towards reduced unfolded rnase b than towards the native protein . similarly , heat - treated soybean agglutinin and urea - treated bovine thyroglobulin were better substrates than the native proteins ( fig5 ). these results demonstrate that the recombinant enzyme has the same specificity for incorrectly folded proteins as the enzyme purified from rat liver . denatured acid phosphatase purified from dt111 is a much better acceptor substrate for rugt than the previously used bovine thyroglobulin and soybean agglutinin . assuming mw of 14 kda , 31 kda and 303 kda for rnase b , sba and tg , acid phosphatase can incorporate 3 h - glucose on a molar basis 130 × better than rnase b , 100 × more than sba and 4 times more than tg . when udp - glucose was used in large excess , and the glucosylation reaction allowed to proceed to completion , the maximum incororation indicated that nearly all 12 glycosylation sites of acid phosphatase are used for glucosylation . this suggested that complete denaturation of acid phosphatase by reduction of its disulfide bonds in the presence of 6m guanidinium hydrochloride ( zapun , 1998 ) followed by desalting prior to glucosylation , did not result in a better incorporation of 3 h - glucose ( fig5 ). thus , it would appear that all of the n - linked glycans in acid phosphatase are recognized and used as targets for glucosylation by rugt . the activity of the four rugt mutants was compared with that of the wild - type enzyme using purified acid phosphatase . the d1334a and d1336a mutants exhibited no activity at all , whereas the n1433a and q1429a mutant showed approximately 2 % and 20 % of the activity of the wild - type enzyme ( fig6 ). the autoradiogram was overexposed to show the weak labeling of acid phosphatase by rugt - q1429a and n1433a . preliminary kinetic characterization of rugt was conducted and its km for udp - glucose and acid phosphatase was determined . in the presence of 0 . 5 μm acid phosphatase , the enzyme exhibited classical michaelis - menten kinetics ( fig7 a ) and it was possible to calculate a km of 44 μm for udp - glucose . a km of 18 μm was previously obtained by trombetta using both soybean agglutinin or bovine thyroglubulin as acceptor substrates for rat liver - purified uggt in 10 mm tris - hcl ph7 . 5 , 10 mm cacl2 ( unpublished results ). however , in the presence of 5 μm udp - glucose , an apparent vmax at 1 . 0 μm acid phosphatase was observed ( fig7 b ). as the acid phosphatase concentration was increased to 2 . 0 and 4 . 0 μm , the rate of glucosylation continued to decrease perhaps indicative of substrate and / or product inhibition . in any case , the apparent km for acid phosphatase was calculated to be 0 . 3 μm . this result could be explained if the acid phosphatase did not get released from rugt following glucosylation . although it has been shown in vivo that association of a secretion - incompetent variant of α1 - antitrypsin to uggt is prevented in the presence of udp - glucose ( choudhury , 1996 ). other characteristics and advantages of the present invention will appear from the following example . the following example is intended to document the invention , without limiting its scope . cloning and expression in insect cells of active rat liver udp - glucose glycoprotein : glucosyltransferase and development of an adequate substrate for this enzyme restriction and modification enzymes were obtained from pharmacia biotech inc ., new england biolabs and mbi fermentas inc . plasmid pfastbac1 , e . coli dh10bac and the 5 ′ race system for rapid amplification of cdna ends ( version 2 . 0 ) were all purchased from gibco / life technologies inc . the marathon cdna amplification kit and marathon - ready cdna were purchased from clontech . plasmids were propagated in e . coli mc1061 ( casadaban , 1980 ) and prepared as described previously ( holmes , 1981 ). oligonucleotides were synthesized on an expedite dna synthesizer ( perseptive biosystems , inc .) and sequencing was done by dideoxy dye termination on an abi prism 377 dna sequencer ( applied biosystems inc .). in order to obtain the coding sequence of the rat uggt , a strategy of cdna “ walking ” was first followed . based on peptide sequence information and a 300 bp stretch of sequence at the 3 ′ end of the gene , oligonucleotide probes were designed and used to screen a rat liver cdna library in lambda zap ( stratagene ). two similar clones of 3 . 3 kb were isolated which contained an open reading frame encoding the c - terminal 385 amino acids , including an er retention sequence and a poly ( a ) tail at the 3 ′ end . the screening of a new rat liver cdna library in λgt10 ( clontech ) with a second probe corresponding to the 5 ′ end of the 3 . 3 kb clone isolated in the first screening gave several overlapping clones . the most interesting one extended the 3 . 3 kb clone further with 1 . 7 kb of sequence from which a third probe was designed . in this third screening , only one positive clone was obtained which extended the open reading frame by 540 bp to 3360 bp . a fourth probe was designed based on this additional sequence information but failed to produce useful clones . subsequently , no full length sequence of the rugt orf were found in any library used . to complete the sequence , the 5 ′ race system was used on rat liver mrnas . the reverse transcriptase reaction was primed with an oligonucleotide corresponding to the 5 ′ end of the 540 bp of sequence information revealed in the third screening . the first strand cdna was later tailed with poly ( dc ) using terminal deoxynucleotidyl transferase . an amplified fragment of 1 . 4 kb was cloned and sequenced . the 5 ′ sequence of this clone consisted of a poly - dg stretch followed by a gc - rich segment of 39 bp preceeding the open reading frame . using this sequence information , rt - pcr was carried out using clontech &# 39 ; s marathon cdna kits to clone the full length cdna as a single fragment of 4 . 6 kb using primers immediately flanking the coding region . two clones from independent reactions were sequenced . for expression , the gene was cloned in the rsrii and kpni sites of pfastbac1 using the following strategy . at the 5 ′ end of the gene , the sequence coding for the first 18 amino acids of rugt was replaced by the sequence coding for the 21 amino acids of the honeybee melittin signal peptide to facilitate the secretion of the protein from baculovirus - infected insect cells ( tessier , 1991 ). at the extreme 3 ′ end of the gene , the sequence coding for the er localization signal heel was replaced by the sequence coding for a terminal ( his ) 6 tag to facilitate purification of the protein ( fig8 ). based on sequence alignments with other nucleoside diphosphate glycosyltransferases , four independent rugt mutants d1334a , d1336a , q1429a and n1433a were designed , amplified by pcr , cloned and sequenced to confirm the presence of diagnostic psti , bsrbi , xbai and naei sites respectively ( fig8 ). recombinant rugt constructs in pfastbac1 were transformed in e . coli dh10bac to produce recombinant bacmids as described by the manufacturer . the presence of the gene was verified by pcr . sf9 insect cells were transfected for 3 days with the recombinant bacmid dna using cellfectin ( gibco / life technologies inc .) as described by the manufacturer to produce stocks of recombinant rugt baculovirus . sf9 insect cells were grown for 3 - 4 days in sf900 ii sfm serum - free medium ( gibco / life technologies inc .) to a density of 2 - 3 × 106 cells / ml in 500 ml micro - carrier spinner flasks ( bellco glass inc .) and infected with { fraction ( 1 / 20 )} of the culture volume of a recombinant rugt baculovirus stock representing an approximate m . o . i . of 2 - 5 pfu / cell . the infected cells were then incubated at 27 ° c . for up to 3 days . aliquots of the infected cells and of the culture supernatant were analyzed on 5 % stacking / 8 % resolving sds - polyacrylamide gels ( sds - page ) followed by western blotting . cells were removed by centrifugation at 3 , 000 × g for 10 minutes . the supernatant medium was collected on ice and adjusted to 40 mm tris - hcl ph 7 . 5 , 0 . 5m nacl . the medium was concentrated approximately 20 fold at 4 ° c . on ym30 membranes ( amicon ) in the presence of 2 mg / ml of the protease inhibitors aprotinin , leupeptin , pepstatin a and e - 64 ( boehringer mannheim canada ). two buffer exchanges were performed using buffer atc / pi ( 40 mm tris - hcl ph 7 . 5 , 0 . 5m nacl , 5 mm cacl2 with protease inhibitors ). the concentrated / diafiltrated medium was centrifuged at 3 , 000 × g for 10 minutes to remove some precipitated proteins and 10 mm imidazole was added to the supernatant before loading twice by gravity flow onto a 1 ml ni ++ − nta superflow column ( qiagen inc .) in a pharmacia fplc c - type column assembly . the ni ++− nta resin had been previously washed with 20 column volumes of ddh2o and equilibrated in buffer atc / pi + 10 mm imidazole . after loading , the column was washed by gravity flow with 20 column volumes of buffer atc / pi + 10 mm imidazole . elution was carried out on a biocad perfusion chromatography workstation ( perseptive biosystems , inc .). the column was first washed with 10 column volumes of buffer atc / pi + 10 mm imidazole at 2 ml / min and proteins eluted with buffer atc / pi + 200 mm imidazole for 20 column volumes at the same flow rate . fractions of 1 ml were collected and aliquots analyzed by sds - page and western blot to confirm the presence of the protein . rugt - containing fractions from the ni ++ − nta column were diluted 10 fold with hq buffer ( 40 mm tris - hcl ph 7 . 5 , 2 mm cacl2 ) before injecting onto a 1 ml poros 20hq column ( perseptive biosystem , inc .) in an pharmacia fplc hr5 / 5 column assembly on a biocad . flow rates for the binding and the elution were set at 1 ml / min while the washes were done at 3 ml / min . the protein was eluted with an nacl gradient of 0 - 750 mm in hq buffer over 30 column volumes . rugt eluted at approximately 350 mm nacl . fractions of 1 ml were collected and aliquots analyzed by sds - page to assess the purity of the protein and to test for rugt activity . the saccharomyces cerevisiae yeast strains used in this study were : w303 - 1a ( mata , ade2 , his3 , leu2 , trp1 , ura3 , can1 ) ( parlati , 1995 ), yns - 7a ( mat a , och1 :: leu2 , mnn1 , his1 , his3 , ura3 ; generous gift from y . jigami ) ( nakayama , 1991 ) and dt111 ( mat a , och1 :: leu2 , mnn1 , mns1 , his1 , his3 , ura3 ). acid phosphatase was produced from dt111 which secretes glycoproteins with asparagine - linked [ glcnac ] 2 -( man ) 9 glycans . dt111 was constructed by disrupting the mns1 gene of yns - 7a using an mns1 :: ura3 cassette ( camirand , 1991 ) from plasmid pbhe5 ( generous gift from a . herscovics ). the ura3 gene was later deleted by selecting for 5 - foa - resistant colonies ( rose , 1990 ). dt111 was grown in ypd medium supplemented with 150 mm kcl as an osmotic stabilizer at 30 ° c . until the o . d . 600 nm reached about 5 . the inoculum was diluted 50 fold in sd — pi medium supplemented with 150 mm kcl and 2 % glucose and grown at 30 ° c . to an o . d . 600 nm of 2 . the sd medium ( rose , 1990 ) was prepared without inorganic phosphate to induce the expression of endogenous acid phosphatase . acid phosphatase was purified from the culture medium by 50 fold concentration / diafiltration against 10 mm sodium acetate ph 5 . 0 followed by anion exchange chromatography on a 1 ml poros 20hq column ( perseptive biosystems , inc .) in a pharmacia hr5 / 5 assembly in 20 mm sodium acetate ph 5 . 0 on a biocad . the flow rates were set at 1 ml / min for the binding and 3 ml / min for the washes . proteins were eluted at 1 ml / min over 30 column volumes using a linear gradient of 0 - 600 mm nacl . acid phosphatase eluted at approximately 100 mm nacl . fractions of 1 ml were collected and aliquots analyzed by sds - page to confirm the presence of the protein . acid phosphatase activity was monitored at o . d . 405 nm following the hydrolysis of 1 mg / ml p - nitrophenyl phosphate ( pnpp ; sigma 104 ) at 37 ° c . for 10 - 60 minutes in 25 mm sodium acetate ph 4 . 0 . acid phosphatase was the major substrate glycoprotein used to determine rugt activity in this study . rnase b was prepared as described elsewhere ( zapun , 1998 ). soybean agglutinin ( sigma ) was denatured by heating at 100 ° c . for 15 minutes . bovine thyroglobulin ( sigma ) was denatured with 8m urea and dialyzed against ddh2o . the acid phosphatase acceptor - substrate (˜ 1 μg ) was mixed with rugt in a 20 μl mixture containing tc buffer ( 10 mm tris - hcl ph 7 . 5 , 10 mm cacl2 ) and 5 μm uridine diphospho - d -[ 6 - 3 h ]- glucose ( 2 - 15 ci / mmol ; amersham life science inc .) and incubated at 37 ° c . for 2 hours . reactions were either analyzed by sds - page followed by fluorography using amplify ( amersham life science inc .) or precipitated with ice - cold 10 % trichloroacetic acid ( tca ), washed twice with tca and the pellets resuspended in 50 μl 1m tris - hcl ph 8 . 0 before counting in a beta counter to quantitate the incorporation of 3 h - glucose in acid phosphatase . for the determination of the km of udp - glucose , 46 nm rugt was mixed with 1 μm of purified acid phosphatase and 5 - 800 μm udp - glucose ( mixture of labeled and unlabeled ) in tc buffer in a final volume of 20 ml . for the determination of the km for acid phosphatase , 10 nm rugt was mixed with 100 μm udp - glucose ( mixture of labeled and unlabeled ) and 0 . 1 - 4 μm of purified acid phosphatase in tc buffer in a final volume of 20 μl . glucosylation reactions were incubated at 37 ° c . and tca precipitated at various times up to 120 minutes to determine the rate of incorporation of 3 h - glucose in acid phosphatase . the 15 mer peptide eekelgtlheeetqe ( amino acid residues 1505 - 1519 ) was synthesized on an 8 - branch map core ( applied biosystems ) on an mps 396 peptide synthesizer ( advanced chemtech ) using fmoc chemistry and desalted on c18 sep - pak reverse phase chromatography cartridges ( millipore / waters ). rabbits were immunized as described by previously ( cooper , 1995 ). periodical bleedings were tested for immunoreactivity to rugt by western blotting and visualized by enhanced chemiluminescence ( ecl ; amersham life science inc .). antibodies generated for this study showed cross - reactivity to two insect cell proteins of about 62 and 70 kda respectively ( fig1 ). while the invention has been described in connection with specific embodiments thereof , it will be understood that it is capable of further modifications and this application is intended to cover any variations , uses , or adaptations of the invention following , in general , the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth , and as follows in the scope of the appended claims . tgcgcggctg cgcggcgggt gacagggctc tgttacaac atg gga ctc ctg att 54 gca ctg gcc tta ctg tgc ctg ttt tcc tta gca gaa gcc aat tca aaa 102 gcc att acc acc tct ctc acc act aag tgg ttt tct gct cca ctg ctg 150 cta gaa gcc agt gag ttc cta gca gaa gac agt caa gag aaa ttt tgg 198 agt ttt gta gaa gcc agt caa aac att gga tca tca gat caa cat gat 246 ser phe val glu ala ser gln asn ile gly ser ser asp gln his asp acc gac cgt tcc tat tat gat gcc ata ttg gaa gct gcg ttt cgg ttc 294 ctg tca cct ctg cag cag aat ttg ttg aag ttt tgt ctc tct ctc cgt 342 tcc tac tca gcc tca att caa gcc ttc cag cag ata gca gtc gac gag 390 cct cca cca gaa gga tgc aag tca ttt ctc tca gtg cat gga aag cag 438 pro pro pro glu gly cys lys ser phe leu ser val his gly lys gln act tgt gat ctg ggc act ctt gag agc ctt ctg ctg act gca cct gac 486 aga cct aaa cct tta ttg ttc aaa gga gat cac aga tat ccc tca tca 534 aat cct gaa agt cca gtg gtc att ttt tat tct gag att ggc cat gaa 582 gaa ttt tct aat att cac cac caa ctt ata tca aaa agc aat gaa gga 630 aaa att aat tat gtg ttc aga cat tat ata tct aat ccc agg aag gag 678 lys ile asn tyr val phe arg his tyr ile ser asn pro arg lys glu ccg gtc cac ctt tct ggc tat ggt gta gaa ttg gcc att aag agc acg 726 pro val his leu ser gly tyr gly val glu leu ala ile lys ser thr gag tac aag gcc aag gat gat act cag gtg aaa ggg acc gag gta aac 774 acc aca gtc att ggg gag aac gat cct att gat gaa gtt cag ggg ttc 822 ctc ttt gga aaa tta aga gaa ctg tac ccc agc ttg gaa gga cag ttg 870 aaa gag ttc cgg aag cat ctc gtg gag agc acc aat gaa atg gcc ccc 918 lys glu phe arg lys his leu val glu ser thr asn glu met ala pro ttg aaa gtc tgg cag ctg caa gac ctc agt ttc cag act gct gcc cgc 966 leu lys val trp gln leu gln asp leu ser phe gln thr ala ala arg atc ttg gct gct cct gtg gag tta gct ctg gtg gtg atg aag gac att 1014 agt cag aac ttt cct acc aaa gcc aga gca ata aca aaa aca gct gtg 1062 ser gln asn phe pro thr lys ala arg ala ile thr lys thr ala val agc gca cag ctt aga gcg gaa gtg gaa gag aac cag aag tat ttc aag 1110 ser ala gln leu arg ala glu val glu glu asn gln lys tyr phe lys gga act ata gga tta cag cct gga gac tca gct ctc ttc atc aac gga 1158 gly thr ile gly leu gln pro gly asp ser ala leu phe ile asn gly ctt cat att gat tta gac acc cag gat atc ttc agt ctg ttt gat act 1206 ctg aga aat gaa gcc cgg gta atg gag ggt cta cat aga cta gga ata 1254 gaa ggc ctt tct cta cat aat att ttg aag ctc aac atc cag ccg tct 1302 gag act gac tac gca gta gac atc agg agt cct gct att tcc tgg gtc 1350 glu thr asp tyr ala val asp ile arg ser pro ala ile ser trp val aac aac cta gag gtt gat agc cga tat aac tca tgg cct tca agt tta 1398 caa gag tta ctt cgc ccc acg ttt cct ggc gtt ata cgg cag atc aga 1446 aag aac cta cat aac atg gtt ttc att gtt gat cct gtt cat gag acc 1494 lys asn leu his asn met val phe ile val asp pro val his glu thr acg gca gag ctg gtt agc ata gcc gag atg ttc ctc agc aat cat ata 1542 thr ala glu leu val ser ile ala glu met phe leu ser asn his ile cca cta agg att ggt ttt atc ttt gtg gtc aat gat tct gaa gat gtt 1590 pro leu arg ile gly phe ile phe val val asn asp ser glu asp val gat ggg atg caa gat gct gga gtc gct gtt ctg aga gca tat aat tat 1638 gtg ggt cag gaa gtg gat ggc tac cat gcc ttc cag act ctc acc cag 1686 val gly gln glu val asp gly tyr his ala phe gln thr leu thr gln atc tac aac aaa gtg agg act gga gaa aag gtg aaa gtt gag cat gtg 1734 gtc agt gtc ttg gag aag aag tac ccg tat gtt gaa gtg aat agc att 1782 ctg ggg att gat tct gct tat gat cag aat cgg aag gaa gcc aga ggc 1830 leu gly ile asp ser ala tyr asp gln asn arg lys glu ala arg gly tac tat gag cag act ggt gta ggc ccc ttg cct gtt gtc ttg ttc aat 1878 ggg atg ccc ttt gaa aag gag cag tta gac ccc gac gag ctg gaa acc 1926 atc aca atg cac aag atc ttg gag acg acc acc ttc ttc caa aga gcc 1974 ile thr met his lys ile leu glu thr thr thr phe phe gln arg ala gtg tat ttg ggt gaa ctg tca cat gat caa gac gtg gta gag tac atc 2022 atg aat cag ccg aat gtt gtt cca aga atc aac tct agg att ttg aca 2070 gct aag cga gag tat ctg gat cta aca gca agc aat aat ttt tat gtg 2118 ala lys arg glu tyr leu asp leu thr ala ser asn asn phe tyr val gat gac ttt gcc aga ttt tct gcc ttg gac tct cgg ggc aag act gct 2166 gct att gcc aac agt atg aac tat ctg aca aaa aaa gga atg tcc tcc 2214 aag gaa atc tat gat gat tcc ttt att agg cca gtg act ttt tgg att 2262 lys glu ile tyr asp asp ser phe ile arg pro val thr phe trp ile gtt gga gat ttt gat agc cct tct ggg cgg cag tta tta tat gac gcc 2310 val gly asp phe asp ser pro ser gly arg gln leu leu tyr asp ala att aaa cat cag aaa acc agt aac aat gtt agg ata agt atg atc aat 2358 aac ccc agc cga gag ata agt gac tca agc acc ccc gtc tcc aga gcc 2406 atc tgg gca gct ctc cag aca cag acc tcc aac tct gct aag aac ttc 2454 atc acc aag atg gtc aaa gag gag acg gca gag gcc ctg gcc gca gga 2502 gtg gac att ggg gaa ttc tct gtc ggg ggc atg gat gtc agt ctt ttt 2550 aaa gag gtc ttt gag tct tcc aga atg gat ttc att ttg tct cat gcc 2598 lys glu val phe glu ser ser arg met asp phe ile leu ser his ala ctg tac tgc agg gat gtt ctg aaa ctg aag aag gga cag aga gtg gtg 2646 atc agc aac gga agg atc att ggg cca ctg gag gac agt gag ctc ttc 2694 aac caa gat gat ttc cac ctc ctg gaa aat atc att ctg aaa aca tcg 2742 asn gln asp asp phe his leu leu glu asn ile ile leu lys thr ser gga cag aaa atc aag tct cat atc caa cag ctt cgc gta gaa gaa gat 2790 gly gln lys ile lys ser his ile gln gln leu arg val glu glu asp gtg gcc agt gat ttg gta atg aag gtg gat gct ctc ctg tca gcg caa 2838 ccc aaa gga gag gcg agg atc gag tac cag ttc ttt gaa gat aag cac 2886 pro lys gly glu ala arg ile glu tyr gln phe phe glu asp lys his agt gca att aaa ctg aag ccc aaa gaa ggg gag aca tac tat gat gtg 2934 ser ala ile lys leu lys pro lys glu gly glu thr tyr tyr asp val gta gct gtt gtc gac cct gtc aca aga gaa gca cag agg ctc gcc ccc 2982 ttg ctc ttg gtt ttg gct cag ctg ata aac atg agt ctg aga gta ttc 3030 atg aat tgc caa tcc aag ctt tcc gac atg cct tta aaa agc ttt tac 3078 met asn cys gln ser lys leu ser asp met pro leu lys ser phe tyr cgt tat gtc tta gag ccg gag att tct ttc act gca gac aac agc ttt 3126 arg tyr val leu glu pro glu ile ser phe thr ala asp asn ser phe gcc aag gga cca ata gca aag ttt ctg gat atg cct cag tct ccg ctg 3174 ala lys gly pro ile ala lys phe leu asp met pro gln ser pro leu ttt act ttg aat ttg aac aca ccc gag agt tgg atg gta gaa tct gtc 3222 aga aca ccc tat gat ctt gat aat att tac cta gaa gag gtg gac agt 3270 arg thr pro tyr asp leu asp asn ile tyr leu glu glu val asp ser ata gtg gct gct gag tat gag ctg gag tat ctg tta ctg gaa ggt cat 3318 tgt tac gac atc acc aca ggc cag ccc cct cga gga ctg cag ttc acg 3366 cys tyr asp ile thr thr gly gln pro pro arg gly leu gln phe thr tta gga act tca gcc aac cca aca act gtg gac aca atc gtg atg gcc 3414 leu gly thr ser ala asn pro thr thr val asp thr ile val met ala aat ctg gga tat ttt cag ctc aaa gcc aac cca gga gcc tgg att ctg 3462 asn leu gly tyr phe gln leu lys ala asn pro gly ala trp ile leu aga ctg agg aag ggg cgc tcg gat gac att tat agg atc tac agc cat 3510 gac gga aca gat tcc cct cct gat gca aat gac gtt gtt gtc atc ctc 3558 aat aac ttc aag agc aag atc atc aaa gtg aag gtt cag aag aag gcc 3606 gac atg gct aat gaa gac ttg ctg agc gac ggg acg aat gag aat gag 3654 tct gga ttc tgg gac tca ttc aag tgg ggc ttc tca gga cag aag act 3702 gag gaa gta aag caa gat aag gac gac ata atc aat att ttc tct gtt 3750 gca tct ggt cat ctc tac gaa agg ttt ctt cgc atc atg atg cta tca 3798 ala ser gly his leu tyr glu arg phe leu arg ile met met leu ser gtc ctg aag aat acc aaa act cct gtg aaa ttc tgg ttc ttg aag aat 3846 tat ttg tcc ccc aca ttt aag gag ttt ata cct tac atg gcc aaa aaa 3894 tyr leu ser pro thr phe lys glu phe ile pro tyr met ala lys lys tac aat ttc cag tat gag ctt gtt cag tac aaa tgg cca cgg tgg ctt 3942 tyr asn phe gln tyr glu leu val gln tyr lys trp pro arg trp leu cac cag cag acc gag aag cag cga att atc tgg ggc tac aag atc ctc 3990 his gln gln thr glu lys gln arg ile ile trp gly tyr lys ile leu ttc ctg gat gtg ctt ttc ccg ttg gtt gtt gac aaa ttc ctc ttt gtg 4038 gat gct gat cag att gtg cgg aca gat ctg aag gag tta aga gat ttc 4086 asp ala asp gln ile val arg thr asp leu lys glu leu arg asp phe aat ttg gat ggt gca cct tac ggt tac acg ccc ttc tgc gac agc agg 4134 asn leu asp gly ala pro tyr gly tyr thr pro phe cys asp ser arg aga gag atg gat ggc tac cgc ttc tgg aag tca ggc tac tgg gcc agt 4182 arg glu met asp gly tyr arg phe trp lys ser gly tyr trp ala ser cat ttg gct gga cga aag tat cac atc agt gcg ctg tat gtc gtg gat 4230 his leu ala gly arg lys tyr his ile ser ala leu tyr val val asp ctg aag aag ttt agg aaa ata gct gct ggt gac aga ctc aga gga cag 4278 tac caa ggt ctg agt cag gat ccc aac agt ctt tca aat ctt gat caa 4326 gat ttg ccc aat aac atg atc cat cag gtg cca atc aaa tcg ctc cct 4374 asp leu pro asn asn met ile his gln val pro ile lys ser leu pro cag gag tgg ctt tgg tgt gag acg tgg tgt gat gat gcc tct aag aag 4422 cgg gcc aag acc atc gac ctg tgt aat aat ccc atg act aag gag ccc 4470 arg ala lys thr ile asp leu cys asn asn pro met thr lys glu pro aaa ctg gag gct gct gtg cgg atc gtc cct gag tgg caa gac tac gac 4518 lys leu glu ala ala val arg ile val pro glu trp gln asp tyr asp cag gag atc aag cag ttg cag acc ctc ttc caa gag gag aaa gag ttg 4566 ggg acc ctg cat gaa gag gag aca cag gaa gga tct cag aag cat gaa 4614 gln glu lys phe trp ser phe val glu ala ser gln asn ile gly ser ser asp gln his asp thr asp arg ser tyr tyr asp ala ile leu glu ile ala val asp glu pro pro pro glu gly cys lys ser phe leu ser val his gly lys gln thr cys asp leu gly thr leu glu ser leu leu lys ser asn glu gly lys ile asn tyr val phe arg his tyr ile ser asn pro arg lys glu pro val his leu ser gly tyr gly val glu leu glu val gln gly phe leu phe gly lys leu arg glu leu tyr pro ser leu glu gly gln leu lys glu phe arg lys his leu val glu ser thr asn glu met ala pro leu lys val trp gln leu gln asp leu ser phe val met lys asp ile ser gln asn phe pro thr lys ala arg ala ile gln lys tyr phe lys gly thr ile gly leu gln pro gly asp ser ala ser leu phe asp thr leu arg asn glu ala arg val met glu gly leu asn ile gln pro ser glu thr asp tyr ala val asp ile arg ser pro ala ile ser trp val asn asn leu glu val asp ser arg tyr asn ser trp pro ser ser leu gln glu leu leu arg pro thr phe pro gly val ile arg gln ile arg lys asn leu his asn met val phe ile val asp pro val his glu thr thr ala glu leu val ser ile ala glu met phe arg ala tyr asn tyr val gly gln glu val asp gly tyr his ala phe gln thr leu thr gln ile tyr asn lys val arg thr gly glu lys val glu val asn ser ile leu gly ile asp ser ala tyr asp gln asn arg lys glu ala arg gly tyr tyr glu gln thr gly val gly pro leu pro val val leu phe asn gly met pro phe glu lys glu gln leu asp pro phe phe gln arg ala val tyr leu gly glu leu ser his asp gln asp arg gly lys thr ala ala ile ala asn ser met asn tyr leu thr lys lys gly met ser ser lys glu ile tyr asp asp ser phe ile arg pro val thr phe trp ile val gly asp phe asp ser pro ser gly arg gln leu leu tyr asp ala ile lys his gln lys thr ser asn asn val arg pro val ser arg ala ile trp ala ala leu gln thr gln thr ser asn ile leu ser his ala leu tyr cys arg asp val leu lys leu lys lys leu leu ser ala gln pro lys gly glu ala arg ile glu tyr gln phe phe glu asp lys his ser ala ile lys leu lys pro lys glu gly glu ser leu arg val phe met asn cys gln ser lys leu ser asp met pro leu lys ser phe tyr arg tyr val leu glu pro glu ile ser phe thr ala asp asn ser phe ala lys gly pro ile ala lys phe leu asp met met val glu ser val arg thr pro tyr asp leu asp asn ile tyr leu leu leu glu gly his cys tyr asp ile thr thr gly gln pro pro arg gly leu gln phe thr leu gly thr ser ala asn pro thr thr val asp thr ile val met ala asn leu gly tyr phe gln leu lys ala asn pro arg ile tyr ser his asp gly thr asp ser pro pro asp ala asn asp val gln lys lys ala asp met ala asn glu asp leu leu ser asp gly asn ile phe ser val ala ser gly his leu tyr glu arg phe leu arg trp phe leu lys asn tyr leu ser pro thr phe lys glu phe ile pro glu leu arg asp phe asn leu asp gly ala pro tyr gly tyr thr pro phe cys asp ser arg arg glu met asp gly tyr arg phe trp lys ser ile lys ser leu pro gln glu trp leu trp cys glu thr trp cys asp asp ala ser lys lys arg ala lys thr ile asp leu cys asn asn pro cggtccgcac aag atg aaa ttc tta gtc aac gtt gca cta gtt ttt atg 49 ccg ttg gtt gtt gac aaa ttc ctc ttt gtg gct gca gat cag att gtg 48 ccg ttg gtt gtt gac aaa ttc ctc ttt gtg gat gcc gct cag att gtg 48 cag gat ccc aac agt ctt tca aat cta gat gca gat ttg ccc aat aac 48 cag gat ccc aac agt ctt tca aat ctt gat caa gat ttg ccg gct aac 48 leu ile asn ala lys trp thr gln thr pro leu tyr leu glu ile ala glu tyr leu ala asp glu gln ala gly leu phe trp asp tyr val ser pro arg ile gln thr his phe gln leu ala glu glu leu arg ser ser cys ser phe asn glu leu gln lys lys leu glu val pro leu ala lys asp ser leu asp ala pro val val thr tyr ser phe asp his ile phe pro gly ser glu asn asn thr arg thr val val leu tyr gly asp leu gly ser ser gln phe arg thr tyr his lys leu leu glu lys glu ala asp lys arg pro val arg leu ser gly tyr gly val glu leu his leu phe asp phe lys val leu lys gln lys his pro thr leu lys arg ala val thr asp gly leu arg ala glu val lys his asn thr glu ala phe gly arg ser leu asn val ala pro pro asp gly ala leu phe ile asn gly leu phe phe asp ala asp thr met asp leu tyr ser leu ile glu ser ser lys lys glu phe ala ile asp ile arg asp thr ala val gln trp val asn asp ile glu asn asp val gln tyr arg arg trp pro ser ser val met asp leu leu arg pro thr phe pro gly met leu arg asn pro thr ala arg ser val ile lys leu ser glu ser phe val ile his gln ala pro ile arg leu gly leu val phe asp ala arg asp ala asn ala ala glu phe ile gln arg leu gly phe gly asp lys glu gln pro ser asn leu gln lys ala val tyr lys gly glu leu thr asp asn asp val ala ile asp tyr leu met asn gln pro his val met pro arg leu asn gln arg ile leu ser gln glu asp val lys tyr leu asp ile asn ser asn arg asp met thr ala thr leu met asp asn leu lys tyr phe leu thr ile trp val phe ala asp leu glu thr asp gln gly arg asp leu leu thr his ala leu asp tyr val gln ser gly glu ser val arg asn leu asn arg leu val trp ala ala met gln ser leu pro pro thr his leu lys met leu arg val tyr ser gln arg val leu gly leu asn lys ser gln arg leu val ile gly asn gly arg leu tyr gly pro leu ser ala gln asp val asn glu glu phe asn ser asp thr leu leu lys ser asp met pro val lys asn phe tyr arg tyr val val glu pro glu val gln phe glu ala asn gly gly arg ser asp gly pro leu ala lys asn ile lys leu thr asp ile gly gly pro val his ser glu phe asp thr leu val asp thr ile val met ala asn leu gly tyr phe gln leu his val val lys leu arg val ser lys lys pro gly met gln gln ala his thr lys ser pro val lys phe trp phe leu lys asn tyr leu ser pro gln phe thr asp phe leu pro his met ala ser glu tyr asn phe thr glu lys gln arg thr ile trp gly tyr lys ile leu phe leu asp ala ile val arg thr asp ile lys glu leu tyr asp met asp leu gly gly ala pro tyr ala tyr thr pro phe cys asp ser arg lys glu met glu gly phe arg phe trp lys gln gly tyr trp arg ser his leu met gly arg arg tyr his ile ser ala leu tyr val val asp leu lys arg asn asn met ile his gln val ala ile lys ser leu pro asp asp trp leu trp cys gln thr trp cys ser asp ser asn phe lys thr ala lys val ile asp leu cys asn asn pro gln thr lys glu ala lys leu thr ala ala gln arg ile val pro glu trp lys asp tyr asp ala glu leu lys thr leu met ser arg ile glu asp his glu asn ser his ser arg met asn leu thr gly leu leu ile phe phe cys his ile ala val leu ala ala leu glu lys lys gly val his thr ser leu lys ala asn trp phe gln gln ile ala val glu tyr gly glu lys cys asp val phe val val val gly glu gln val ser cys glu tyr thr lys leu glu lys met ile lys asp ala lys thr asn ser gln val leu glu ser asp his ile pro val ser leu ser gly tyr gly val glu leu ala ile lys asn thr his pro asp ser val asp ala ile glu ser phe arg val asn leu lys ser tyr gln ala ala gln lys ile val asn ala gly pro ala asp ala ile gly thr leu glu glu tyr ser gln asn phe pro thr his ala arg gln asn arg lys met leu glu lys ala ser ile glu val gly glu thr phe his ser met gly ile asn arg glu tyr leu ser ile leu val gly met asp thr ser asp asp glu lys thr thr tyr ala val asp his arg glu gly tyr pro phe phe ile asn asn leu asp thr asp lys lys tyr lys gln trp gly asn ser val lys leu met leu gln pro tyr tyr pro gly met ile arg pro ile ala arg asn leu phe ser leu val phe val val asp pro ser thr ser glu gly arg lys phe leu arg ile gly gln thr phe asn ser his asp ile ala met arg ile gly tyr ile phe ala val asn gln asp thr lys ala ser gly glu thr asp leu gly val ala arg lys his gly phe glu phe val gln lys thr gly leu asn ser ala pro lys val leu leu asn gly phe ile leu asp asp glu gly val arg pro lys ile gln arg ala ile met glu gly lys leu thr asp arg met asn val gly asn trp val leu glu gln lys asp val met pro arg ile asn lys arg ile leu ser ala pro ser lys lys thr tyr val glu ile asp ala glu ala ala ser gly arg arg phe ile tyr asn ser leu gln ile leu lys asn ser ala asn ser arg val gly ile ile phe asn pro leu lys leu ser asn glu glu tyr ala ala asp phe ile ser gly lys ile thr phe asp asp leu ser val gly gly met asp thr ala lys phe val gly asn ala leu gln val gly pro leu glu ser ser glu his phe ala glu val ile ser ser his leu lys lys trp glu phe asp val ser lys his ala ser ser gln lys arg thr trp val ser ile gln gly asp glu his ser val val thr leu pro ala asp glu met asp arg pro ala leu gly ser ile leu his leu ile lys lys val thr asn cys glu ile lys ile val met asn pro lys asp lys his ser glu leu pro leu lys gly asn leu asn thr asn val val arg phe asp asn leu pro ser lys gln leu leu thr leu ser leu gln ala pro asp ser trp ile val glu ala val ser ala lys tyr asp leu asp asn ile lys met glu gln ala asn gly asp val thr ala glu phe ala leu gln his leu leu leu asp gly gln cys phe asp glu val ser gly gln pro pro arg gly leu gln phe thr leu gly thr asp lys asn pro lys gln phe asp thr ile val met ala asn leu gly tyr phe gln leu lys ala asn pro gly ala trp ser his val gly ala glu lys ile gly glu asp val leu gln val val val ile asn val phe ser leu ala ser gly his leu tyr glu arg phe lys phe trp leu leu lys asn tyr leu ser pro gln phe lys glu thr leu pro thr leu ala lys his tyr gly phe glu tyr glu leu ile glu leu met glu leu met lys phe asp leu gly asn ala pro tyr gly tyr val pro phe cys glu ser arg lys glu met asp gly phe arg phe trp lys gln gly tyr trp ala asn his leu ala gly arg arg tyr his ile ser ala leu tyr val ile asp leu gln lys phe arg gln ile ala ala val lys ile lys ser leu pro gln glu trp leu trp cys glu thr trp asn pro leu thr lys glu pro lys leu asp ser ala ala arg ile ile gly glu trp lys thr tyr asp asp glu ile arg glu val ile ser gly tyr ala ala lys pro leu asp val lys ile ala ala thr phe asn ala glu gly phe ile trp tyr leu asn his leu ser asp leu leu asp ala arg asp leu ile pro phe his glu leu tyr tyr lys leu ala leu glu asn glu arg leu phe gly met thr ser asp ser leu gln thr val thr pro ile tyr ala his tyr leu ser ile gln pro asp val ser asn his leu ile glu glu leu asn gln phe gln ser gln tyr val pro glu gly asp arg phe glu ala leu gly ile lys ser ser lys val leu asp ile gly lys gln leu his thr val ile tyr pro ile phe pro ser ser pro ser pro val gln thr gly met val cys ala ala asn asp asp asp glu phe ala gln thr val cys lys ser phe phe tyr ile ser lys glu ser gly thr asp ser ala leu lys phe leu tyr lys cys leu asn ser asp ser phe phe asp his tyr met ser lys ser asn ser trp val asn arg ile ser his asp glu asn tyr asp arg ser met tyr gly ile phe leu asp ile lys arg val leu glu asn val gly ser leu asn his glu asp ile leu leu ile gly ser ser asn ala lys tyr ser phe trp leu val asp pro ser ser gly val val asn asp met asp lys ala leu lys phe cys lys ser ala leu leu leu asn gly arg met ile cys ser phe ser val asp ser leu asn thr ala asp leu lys met leu met gln met glu lys leu phe pro arg asp phe ile tyr asn lys leu gly val gly asn leu glu ala val ser lys leu asn gly val gly val arg ile his leu asn pro lys gln thr leu ser glu leu pro leu thr arg phe tyr arg tyr ser ile ser ala glu pro glu phe asp ala leu gly his leu glu glu ser tyr val glu phe asp asn leu pro ala asp thr leu leu thr met asp ile glu ala arg asp ala trp thr val met gln lys asp val asp ile asp leu phe asn ile lys leu glu his thr ser glu ala glu ala leu asp ser his thr ala ile tyr glu leu lys asn ile leu val gln leu lys leu gly asn leu thr asn ser his val thr asp thr ile val leu ser asn leu gly tyr phe gln leu lys ala asn pro gly val trp thr leu glu pro met asp gly arg ser ser gln phe tyr glu ile asp ser phe glu gly val thr leu asn pro val met arg arg lys pro gly phe glu ser ala asp ile met asp glu asp leu ser ser his lys lys glu ala ser ile asn ile phe ser val ala ser gly his leu tyr glu arg phe leu tyr ile met thr lys ser val ile glu his thr asp lys lys val lys phe trp phe ile glu asn phe leu ser pro cys phe tyr ile thr tyr asn trp pro his trp leu arg lys gln glu glu lys gln arg glu ile trp gly tyr lys ile leu phe leu asp val leu phe pro leu glu leu his lys val ile tyr val asp ala gln ile val arg gly tyr thr pro met cys asp ser arg glu glu met glu gly phe arg his ile ser ala leu tyr val val asp leu asp arg phe arg lys met his leu ile pro ile tyr ser leu pro gln asp trp leu trp cys glu cys gln asn pro leu thr lys glu lys lys leu asp arg ala arg arg gln val ser glu trp thr ser tyr asp asn glu ile ala ser val leu ala trp thr tyr ser leu arg tyr gly ile pro glu ser ala gln val pro met gly met ile gln his asp ile ser ser asn ala glu gln asp asp ala asn ser ser tyr phe val leu asn gly asn arg tyr glu lys pro asp asp val phe tyr leu lys ser lys asp leu thr ile gln gln thr asn ser glu ala pro ile leu ile leu tyr gly cys pro thr val met asn gly glu gly lys phe arg phe ile trp arg ser thr cys ser leu asp gly lys ser val glu tyr pro leu thr his pro leu glu ile thr leu gln asn gly ser arg met ser ser ile pro gln leu lys lys ile leu tyr thr val pro lys glu ile leu val gly ala asp asn asp arg val thr ser leu ile ser glu phe tyr gln tyr lys lys asp ile thr ser asn glu glu leu asn ser lys gly phe asp tyr asn met leu gly leu tyr ile asn gly gln asn trp lys ile thr ser leu thr pro ile thr asn leu leu gln glu leu glu pro ser lys cys ile leu asp gln asn leu gln pro ile lys met asp leu his thr ile pro gly phe gly glu leu pro glu ile lys gln asn trp ser glu ile ile phe val ile asp phe ala arg leu glu asp ser glu val lys glu ala leu gly pro phe arg lys asn trp asn tyr leu ile ala lys val ile lys lys asp thr glu phe ile arg lys glu leu ser asn ser ser pro lys asn lys gln ile ser val arg asp leu leu his tyr lys ser ala asn leu arg his asn lys tyr thr pro asn tyr phe ala asp ser val tyr ser ser val asn asn thr ala leu glu ser val cys ser glu arg ile gly val asp asp phe gly ser ile his ala leu lys arg leu arg asn leu asp pro ile gly ser ile asn thr phe ile asp ala leu lys leu lys leu his lys trp leu pro asp ile pro leu phe glu leu gln lys gly leu arg thr ile asp ser val phe ala leu asp leu leu phe pro gly phe phe arg pro asn asn leu thr met phe glu asp gly lys ser ala met ile leu ser leu val glu gln phe arg pro leu lys phe val asn glu leu his ser phe ser ser lys thr val leu ser thr gly asn ile his thr asp lys phe leu lys gly cys tyr ile lys ser cys asp ser arg tyr thr val gln ser phe ser thr asp gly his pro asp phe ile lys cys pro glu thr gln lys val asn phe phe ile leu asp gln pro phe ile ser asp thr leu arg lys ser cys glu tyr ile asn ser ser asp glu met arg gly asn val ile phe leu asn tyr glu trp pro gln tyr met ser pro thr glu val pro leu asp pro phe asp ile phe gln arg phe arg glu leu asp ala gly asp lys tyr arg ile his tyr gln arg ile ser thr asp ala met ser leu val asn ile gly gln asp leu val asn asn leu gln leu glu val pro ile arg phe leu lys gly ser tyr lys lys lys leu val ile asn asp glu cys val ser glu trp lys lys lys ile asn lys phe ala ser ser pro gly asp glu asp val pro gly glu ser val ser ser lys tyr gln asp ser asp asn ala ala pro his leu tyr glu arg phe leu arg ile met xaa xaa ser val leu lys tyr asn phe xaa tyr glu leu ile xaa xaa xaa tyr lys trp pro arg phe cys asp ser arg xaa glu met asp gly phe arg phe trp lys xaa ile ser ala leu tyr val val asp leu xaa arg phe arg lys ile ala tyr gly tyr thr pro phe cys asp ser arg arg glu met asp gly tyr arg phe trp lys thr gly tyr trp ala ser his leu leu arg arg lys tyr his ile ser ala leu tyr val val asp leu lys lys phe arg arg pro leu ser leu glu lys val ile phe val asp ala asp gln ile ile arg xaa asp met gly glu leu tyr asp met asp ile lys gly arg pro leu ala tyr thr pro phe cys asp asn asn arg xaa met asp gly tyr pro leu ser 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