the enzyme 1,3-galactosyltransferase ( galt ) catalyzes the binding of 1,3galactose ( gal ) on n - acetyllactosamine ( gal1,4glcnac ) to produce gal1,3gal1,4glcnac - r ( -gal epitopes ) on the cell surface of almost all mammals , but not on those of humans , apes , and old world monkeys . several research groups have produced 1,3-galactosyltransferase ( ggta1 ) gene knockout ( galt ko ) pigs in order to overcome the problem of immune rejection after xenotransplantation [ 36 ] . organs from these pigs avoid both hyperacute and acute humoral xenograft rejection without requiring complement inhibition or antibody absorption . although galt ko - derived organs prolong xenograft survival in recipients , xenografted organs from these animals result in progressive organ death . carbohydrates such as hanganutziu - deicher ( h - d ) , thomsen - friedenreich ( t or tf ) , tn , and sialyl - tn play a pivotal role in the acute immune rejection of pig xenografts . h - d antigens are glycoconjugate - bound n - glycolylneuraminic acids ( neu5gc ) which are a type of sialic acid ( sia ) , as is n - acetylneuraminic acid ( neu5ac ) . in cells , neu5gc is mainly produced from neu5ac by the catalyst cmp - n - acetylneuraminic acid hydroxylase ( cmah ) with the cofactors cytochrome b5 and nadh [ 10 , 11 ] . in this study , we tested whether sia - containing glycoconjugate expression in pigs could be altered by deficiency of the ggta1 gene . to accomplish this , we examined whether increased production of neu5gc seen in heterozygote galt ko pigs was caused by increased expression of 2,6-sialyltransferase ( 2,6st ) and the cmah cofactor nad - isocitrate dehydrogenase ( idh ) . in this study , we used 3 control and 3 galt heterozygote ko pigs ranging in age from 4 to 6 weeks . the treatment of the pigs used in this research followed the guidelines set by the national institute of animal science 's institutional animal care and use committee , suwon , republic of korea ( approval no . control and heterozygote galt ko livers were minced with a tissue grinder under liquid nitrogen . the organ powders were washed twice with phosphate buffered saline ( pbs ) and then centrifuged at 1,500  g for 10  min . the pelleted organ powders were resuspended in 20  mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ; ph 7.4 ) containing 0.5% nonidet p-40 , protease inhibitor cocktail ( roche , almere , netherlands ) , and lysed by sonication . protein concentrations were determined using a bicinchoninic acid ( bca ) protein assay kit ( pierce , rockford , il , usa ) with a bovine serum albumin standard . total rna was extracted from control and heterozygote galt ko liver tissue using a micro - to - midi total rna purification system ( invitrogen , la jolla , ca , usa ) . real - time reverse transcriptase - polymerase chain reaction ( rt - pcr ) was conducted using a dna engine chromo4 system ( bio - rad , hercules , ca , usa ) and sybr green as the double - stranded dna - specific fluorescent dye ( bio - rad , hercules , ca , usa ) . we used pig h2a histone family , member z ( ph2afz ) as an internal standard to normalize the rt - pcr reaction efficiency and to quantify in heterozygote galt ko pig- and control - derived liver mrna . after normalization with ph2afz mrna , we compared the relative expression of each mrna in the heterozygote galt ko pig - derived liver genes with those of the controls . data are presented as the mean of the gene expression measurements for each individual control and heterozygote galt ko liver sample ( table 1 ) . the 1,3-galactosyltransferase ( galt ) , 2,3- and 2,6-sialyltransferase ( 2,3st and 2,6st ) activity were assayed as previously described with minor modifications [ 12 , 13 ] . in brief , an acceptor substrate , lacto - n - neotetraose ( lnnt ; sigma - aldrich ) was labeled with 2-aminobenzamide ( 2-ab ) . the mixture was comprised of 2  mg of lnnt , 0.2  mg of 2-ab , 0.24  mg of sodium cyanoborohydride , 6  l of acetic acid , and 14  l of dimethyl sulfoxide ( dmso ) . the mixture was incubated at 65c for 3  h and then was purified using glycoclean s cartridges ( prozyme , hayward , ca , usa ) . the assay mixture for galt activity contained 20  mm hepes buffer ( ph 7.2 ) , 0.25% nonidet p-40 , 10  mm mncl2 , 33  mm nacl , 3  mm kcl , 20  mm udp - galactose , 200  mm galactose , and 100  m acceptor substrate ( lnnt - ab ) , and 6  l of organ lysate for a total volume of 20  l . the assay mixture for 2,3st and 2,6st activity contained 20  mm hepes buffer ( ph 7.2 ) , 0.25% nonidet p-40 , 10  mm mncl2 , 33  mm nacl , 3  mm kcl , 20  mm cmp - neu5ac , 200  mm galactose , and 1  mm acceptor substrate ( lnnt - ab ) , and 6  l of organ lysate for a total volume of 20  l . after incubation at 37c for 6  h , 80  l of water was added to each sample mixture and the reaction was terminated by boiling for 5  min , followed by centrifugation of the samples at 15,000  g for 10  min . the resulting supernatant was analyzed by reverse - phase high - performance liquid chromatography ( rp - hplc ) using an octadecyl silane ( ods ) column ( 4.6  150  mm , tsk - gel column ods-80tm ; tosoh bioscience , tokyo , japan ) . the products and substrate were isocratically separated with 20  mm ammonium acetate buffer ( ph 4.0 ) containing 0.15% n - butanol at 55c . each peak was detected with a fluorescence detector ( model rf-10a ; shimadzu , tokyo , japan ) at excitation and emission wavelengths of 330 and 420  nm , respectively . we defined enzyme activity as picomoles of product per hour per milligram of organ lysate protein . control and heterozygote galt ko livers were also tested by ella , using griffonia simplicifolia isolectins b4 ( gs - ib4 ) , maackia amurensis agglutinin ( maa ) , and sambucus nigra agglutinin ( sna ) . a 50  l sample of organ lysate ( 25  g protein / well ) was diluted in pbs , dispensed into 96-well microtiter plates and incubated at room temperature for 2  h. the organ lysates were then dispersed and washed once with pbs containing 0.1% tween 20 ( pbst ) and blocked with pbst containing 2% bovine serum albumin . biotinylated gs - ib4 , maa , and sna solutions ( all 100  l at 0.1  g / ml ) were applied and incubated at room temperature for 2  h. the samples were washed 3 more times with pbst and incubated for another 2  h with 100  l of horseradish peroxidase - conjugated hen egg white avidin ( 0.1  g / ml ) . the reaction was developed using o - phenylenediamine dihydrochloride ( pierce , rockford , il , usa ) according to the manufacturer 's instructions . absorbance was measured at 490  nm using a multiskan fc microplate photometer ( thermo scientific , pittsburgh , pa , usa ) and signal levels were normalized using actin . the 2-dimensional gel electrophoresis ( 2de ) and spot analysis were performed as previously described , with slight modifications . total proteins ( 500  g ) for analytical runs were transferred into ipg strip holder channels ( bio - rad , hercules , ca , usa ) . the 2de process separates protein mixtures by ief ( ph 310 ) in the first dimension and sds - page ( 7.517.5% linear gradient ) in the second dimension . target spots , identified using pdquest software ( bio - rad , hercules , ca , usa ) , were excised from the gel , destained , and subjected to in - gel digestion with bovine trypsin ( roche , almere , netherlands ) . we created a match set consisting of 6 images , 3 from control and 3 from heterozygote galt ko livers . one of the control images was selected as the match set standard for spot matching . the protein abundance of detected spots was quantified and normalized by dividing the optical density ( od ) values of individual spots by the total od values of all spots present in the images . peptides were then analyzed by matrix assisted laser desorption ionization time - of - flight mass spectrometry ( maldi - tof ms ) as described previously . briefly , trypsin digestion reactions were terminated with trifluoroacetic acid ( tfa ) at a final concentration of 10% . peptides were concentrated and desalted using ziptipm - c18 ( millipore , etten - leur , netherlands ) and eluted directly onto the maldi target in 1  ml of a saturated solution of -cyano-4-hydroxycinnamic acid ( chca ) in 50% acetonitrile . peptides were analyzed using a voyager de - str maldi - tof mass spectrometer ( applied biosystems , framingham , ma , usa ) in reflection mode , at an accelerating voltage of 20  kv . database searches were performed using protein prospector ( http://propector.ucsf.edu ) and prowl ( http://www.proteometrics.com ) . real - time rt - pcr in figure 1 , glycosyltransferase activity assay in figure 2 , and ella in table 2 were analyzed using 3 controls and 3 heterozygote galt ko pigs . the sialyltransferase family is generally classified into 4 different subfamilies , st3gal , st6gal , st6galnac , and st8sia , according to the carbohydrate linkages synthesized . we examined how deficiency of ggta1 gene changed the sialyltransferase mrna expression in heterozygote galt ko liver . we used ph2afz as an internal standard to normalize the rt - pcr reaction efficiency and to quantify in heterozygote galt ko pig- and control - derived liver mrna . after normalization with ph2afz mrna , the mrna expression of heterozygote galt ko pig- and control - derived liver genes showed that st3gal 1 , st3gal 3 , and st6gal 1 gene expressions were upregulated ( 1.55-fold , p < 0.001 ; 1.13-fold , p < 0.05 ; 3.16-fold , p < 0.001 , resp . ) , whereas st6galnac 5 was downregulated ( 0.49-fold , p < 0.001 ) in heterozygote galt ko liver compared to the control ( figure 1 ) . this suggests heterozygote galt ko pig - derived organs exhibit a higher sia - containing glycoconjugate on glycoprotein and glycolipid than controls , indicating that they act as an immune antigen in allo- or xeno - grafted organs . as shown in figure 2 , galt activity in heterozygote galt ko liver was significantly lower than in control liver , whereas 2,3 and 2,6st activity was significantly higher than in controls . it is especially interesting that mrna expression in heterozygote galt ko liver mirror protein activity . as we expected , ella analysis showed that optical density of gs - ib4 for -gal epitope in heterozygote galt ko liver ( 2.1138  0.1864 , p < 0.001 ) was significantly lower than that of the control ( 3.3282  0.1775 ) . however , there were significantly more sia - containing glycoconjugate epitopes in galt ko liver than in controls . < 0.05 ) in heterozygote galt ko liver was significantly higher than those of the control ( 3.5984  0.1192 and 20.0019  2.2285 , resp . ; these results suggest that 2,6st may preferentially use neu5gc rather than neu5ac as a donor substrate so that heterozygote galt ko pigs have more glycoconjugate - bound neu5gc epitopes than controls . to examine whether increased production of neu5gc in heterozygote galt ko pigs was caused directly by increased expression of cmah , we tested whether the expression of full - length cmah or variant mrna in pig liver could be altered by deficiency of the ggta1 gene . the full length of pig cmah cdna and its splicing isoform , variant 3 , showed constant expression levels , whereas the cmah variant 2 in heterozygote galt ko livers was greater than that of controls ( figure 3 ) . this suggests that cmah variant 2 mrna in heterozygote galt ko liver may be involved in the underlying conversion mechanism from neu5ac to neu5gc . finally , we compared the proteomes of heterozygote galt ko liver with control - derived liver by 2de analysis ( figure 4 ) . heterozygote galt ko livers showed a higher expression of nad - isocitrate dehydrogenase ( idh ) , compared to those of controls ( table 3 ) . idh converts nad to nadh in the tca cycle , and cmah enzyme uses nadh as a cofactor for a hydrogen source in the catalytic reaction of cmp - neu5ac to cmp - neu5gc . these data suggest that neu5gc accumulation in heterozygote galt ko pig - derived organs may be caused by an increase of 2,6st and cmah activity . sias are typically found at the terminal ends of oligosaccharide chains , which are involved in various biological processes , such as immune - response , inflammation , and tumor cell metastasis [ 1719 ] . it is well - known that st3gal 1 and st3gal 4 catalyze the binding of 2,3-sialic acid on gal1,3galnac - r to produce sia2,3gal1,3galnac - r , whereas st3gal 3 and st6gal 1 catalyze the binding of 2,3- and 2,6-sialic acid on gal1,4glcnac - r to produce sia2,3gal1,4glcnac - r and sia2,6gal1,4glcnac - r , respectively . additionally , st6galnac 5 catalyzes the binding of 2,6-sialic acid on sia2,3gal1,3galnac - r to produce sia2,3gal1,3(sia2,6)galnac - r . as shown in figure 1 , st3gal 1 , st3gal 3 , and st6gal 1 were upregulated in galt ko pig liver cells as compared to controls . similarly , both 2,3st and 2,6st were increased in heterozygote galt ko livers compared to control livers . therefore , upregulated st3gal 1 , st3gal 3 , and st6gal 1 may increase sia2,3gal1,3galnac - r , sia2,3gal1,4glcnac - r and sia2,6gal1,4glcnac - r , respectively , on glycolipid and glycoprotein within heterozygote galt ko livers . maa consists of 2 molecular species , a hemagglutinating hemagglutinin ( mah ) and a mitogenic leukoagglutinin ( mal ) . both isolectins are able to interact with sialic - acid - contained glycoconjugates ; mah has higher affinity toward sia2,3gal1,3(sia2,6)galnac on o - glycan , but mal preferentially binds to the sia2,3gal1,4glcnac structures of n - glycan chains [ 20 , 21 ] . sna lectin , however , specifically binds to sia2,6gal / galnac structures of n- or o - glycan chains . as expected , the signal level of maa in heterozygote galt ko liver was higher than that of the controls ( table 2 ) . this result is reasonable because the decrease of galt activity in heterozygote galt ko liver , as compared to control livers , results in an increase in the nonreducing end ( gal1,4glcnac - r ) of glycan chains . however , the sna signal level in heterozygote galt ko liver was similar to that of control liver . this may be explained by the decreased expression level of st6galnac5 in the heterozygote galt ko liver as compared to controls ( figure 1 ) . the signal intensity of sna toward sia2,6-containing glycoconjugates might be attenuated with a decrease in availability of sia2,6galnac . reported that ( 1 ) galt ko mice showed only a modest increase in n - acetyllactosamine residues and exhibited little sialylation and ( 2 ) overexpression of h substance and suppression of the -gal epitope in htf mice were associated with a marked reduction in 2,3-sialylation and exposure of normally cryptic antigens such as sialylated tn and forssman antigens . pigs differ from mice , however , in that pigs have a 10- to 100-fold higher expression of -gal epitopes than mice have . additionally , galt ko pigs showed up - regulation of sialylated epitopes compared to the nontransgenic wild type pigs . it is not appropriate to compare lectin binding in pigs to lectin binding in mice because mice and pigs exhibit markedly different glycosyltransferase expression . in our study , galt ko pigs showed up - regulation of 2,3- and 2,6-sialyltranferase compared to the control . whereas mice splenocytes have cryptic epitopes in the inner cell , we used liver lysates in ella . all glycan epitopes of the organ are exposed within lysates and lectin ( maa and sna ) can directly bind to their sialylated epitopes . this study demonstrates that galt ko pig has higher 2,3- and 2,6-sialylation when compared with those reported for galt ko mice . when cmp - neu5ac and cmp - neu5gc were compared as donor substrates , st6gal 1 showed 47 times greater activity toward cmp - neu5gc than cmp - neu5ac , whereas there was no significant difference between the activity of st3gal 1 toward these 2 substrates irrespective of the origin of the enzymes . similarly , the high level of st6gal 1 in heterozygote galt ko liver may preferentially transfer neu5gc to the nonreducing galactose residue in glycan chains . these results indicate that a deficiency of galt moderately increased 2,3-linked neu5gc glycoconjugates , but highly increased 2,6-linked neu5gc glycoconjugates . nad - related isocitrate dehydrogenase , also known as idh , is an enzyme that participates in the citric acid cycle . it catalyzes the third step of the citric acid cycle , the oxidative decarboxylation of isocitrate , producing -ketoglutarate and co2 while converting nad to nadh . in order to produce neu5gc from neu5ac the heterozygote galt ko liver showed up - regulation of nad - related isocitrate dehydrogenase alpha subunits , compared to control liver ( table 3 ) . the longest 1734  bp form encodes 577 amino acids and is designated as  full length  . the shorter 1125 and 1056  bp forms designated as  variant 2  and  variant 3  , have an in - frame stop codon and encode 374 and 351 amino acids , respectively . however , it remains unknown whether the cmah - derived splicing isoforms have enzyme activity . as shown in figure 3 , the amount of variant 2 mrna in galt ko pigs was more significant than in controls , as determined by real - time rt - pcr . these observations suggest that cmah variant 2 mrna in heterozygote galt ko liver may be involved in the underlying conversion mechanism from neu5ac to neu5gc . in the present study , we observed a clear decrease in galt activity and increase in neu5gc content in heterozygote galt ko pigs as compared to controls . in conclusion , neu5gc accumulation in heterozygote galt ko pig - derived organs may be caused by a preference for neu5gc over neu5ac as a donor substrate due to upregulated 2,6st and cmah activity . thus , the deletion of both the cmah and ggta1 genes in pigs is expected to further prolong xenograft survival . even though heterozygote galt ko pigs successfully produced fertilized sperm , the gnotobiotic facility in our system was limited . therefore , while we were unable to acquire them , homozygote pigs might provide further clues to the long - standing question of why galt ko - derived pig organs transplanted to baboon result in acute rejection at 179 days after transplantation .
we examined whether deficiency of the ggta1 gene in pigs altered the expression of several glycosyltransferase genes . 
 real - time rt - pcr and glycosyltransferase activity showed that 2 sialyltransferases [ 2,3-sialyltransferase ( 2,3st ) and 2,6-sialyltransferase ( 2,6st ) ] in the heterozygote galt ko liver have higher expression levels and activities compared to controls . 
 enzyme - linked lectin assays indicated that there were also more sialic acid - containing glycoconjugate epitopes in galt ko livers than in controls . 
 the elevated level of sialic - acid - containing glycoconjugate epitopes was due to the low level of -gal in heterozygote galt ko livers . 
 furthermore , proteomics analysis showed that heterozygote galt ko pigs had a higher expression of nad+-isocitrate dehydrogenase ( idh ) , which is related to the cmp - n - acetylneuraminic acid hydroxylase ( cmah ) enzyme reaction . 
 these findings suggest the deficiency of ggta1 gene in pigs results in increased production of n - glycolylneuraminic acid ( neu5gc ) due to an increase of 2,6-sialyltransferase and a cmah cofactor , nad+-idh . 
 this indicates that neu5gc may be a critical xenoantigen . 
 the deletion of the cmah gene in the galt ko background is expected to further prolong xenograft survival .