glycosylation plays an important role in several types of recognition processes ranging from fertilization and development to pathological events and cell death . in earlier times of glycobiology the role of o - glycans was merely seen in the influence of physical parameters , like ensuring protein stability and tertiary structure , serving as  stalks  in membrane bound receptors and providing the basis for rigid conformation of the polypeptide backbone ; the more specialized functions had been allocated to n - glycans . in the meantime it has been shown , that o - glycans can be also relevant for the fine tuning of biological processes , such as modulation of enzyme activity , they may act as signal molecules or sorting determinants [ 15 ] . mucin - type o - glycosylation is the most frequent type of o - glycosylation , present in all kinds of animals containing an n - acetylgalactosamine as the linkage sugar to a hydroxy amino acid of a peptide [ 68 ] . this precursor is further elongated by adding galactoses , n - acetylglucosamines , n - acetylgalactosamines , fucoses , sialic acids and other sugars depending on the organism , tissue and developmental stage . the first enzyme involved in the biosynthesis of these o - glycans is a member of an evolutionarily highly conserved family of udp - galnac : polypeptide galnac transferases ( ppgalnacts , [ ec 2.4.1.41 ] ) , which transfer the galnac residue from udp - galnac to a serine ( ser ) or threonine ( thr ) of a polypeptide chain . the family members share a common structure consisting of a short transmembrane domain , a stem region , a gal / galnac motif , a manganese binding site and , quite commonly , a ricin - like lectin domain . small differences in structure seem to be related to tissue distribution and acceptor specificity . in humans 20 representatives of this enzyme family have been found , 19 in rodents , 14 in drosophila melanogaster , 9 in caenorhabditis elegans and several in other organisms [ 911 ] . the large number of seemingly redundant homologues of the same enzyme in one organism indicates that it requires a reliable backup system . in most of the experiments in mice the loss of a single galnact - gene caused no obvious phenotype ( for a review see 10 ) . the ppgalnacts have been clustered into groups and subgroups by their primary structure . while n - glycosylation is restricted to asn - residues within the consensus sequence asn - x - ser / thr , for mucin - type o - glycosylation no strict amino acid sequence can be determined . however , a number of studies on vertebrate enzymes demonstrate that the different groups and subgroups favour specific amino acids close to the glycosylation site of the acceptor peptide . furthermore these groups differ also in their ability to transfer galnac - residues to already glycosylated acceptor substrates and their expression levels vary for different tissues . molluscs are highly successful in survival , are able to adapt to changing environments and are intermediate hosts of some parasites . they combine glycosylation features of mammals , worms and insects which makes them an interesting model for studies of biosynthetic pathways , in particular glycosylation processes . ppgalnact from biomphalaria glabrata ( genbank : kc18251 ) , so far the only cloned and characterized glycosyltransferase from mollusc origin , is a 600 amino acid type ii membrane protein containing all the above mentioned structural domains . it is a member of group ib being a typical t2 enzyme , with a ph optimum at 6.06.5 , dependence on divalent cations and it is able to glycosylate non- as well as multi - glycosylated acceptor peptides . here we present a detailed evaluation of the snail ppgalnact donor and acceptor preferences and elucidate the order and position of the glycosylated amino acids in case of multi - glycosylation . furthermore , the influence of the lectin domain on the specificity of the snail enzyme is revealed . spodoptera frugiperda cells ( sf9 , atcc crl-1711 ) were cultivated in ipl41 medium ( safc biosciences , st . louis , usa ) containing yeast extract , a lipid mixture supplemented with 10  % fetal calf serum , at 27  c . acceptor peptides were obtained from cellmano biotech co. , ltd . , shanghai , china ( table  1).table 1acceptor peptides used in this studynamesequencemuc1aappahgvtsapdtrpapgcmuc1aahgvtsapdtr ( short version of muc1a)muc2ptttpitttttvtptptptgtqtkmuc5acgttpspvpttsttsapcht 1appahpgp 
 t 
 pgprpapg 
 cht 2appahpgv 
 t 
 pgprpapgcht 3appahpgp 
 t 
 pgyrpapgcht 4appahpgp 
 t 
 pgkrpapgcht 5appahpgp 
 t 
 pghrpapgcht 6appahpgv 
 t 
 pgyrpapgcht 7appahpgi 
 t 
 pgprpapgcht 8appahpgf 
 t 
 pgprpapgcht 9appahpgp 
 t 
 egprpapgcht 10appahpgp 
 t 
 igprpapgcht 11appahpgp 
 t 
 prprpapgcht 12appahhgp 
 t 
 pgprpapgcht 13appahpvp 
 t 
 pgprpapgcht 14appahpgl 
 t 
 pgprpapgcht 15appahhvv 
 t 
 eryrpapgcht 16appahdfv 
 t 
 paprpapgcht 17appahdfp 
 t 
 paprpapgcht 18appahpfv 
 t 
 paprpapgcht 19appahifv 
 t 
 paprpapgcht 20appahvfv 
 t 
 paprpapgcht 21appahdgv 
 t 
 paprpapgcht 22appahpgp 
 t 
 paprpapgcht 23pssspisssssvspspspsgsqsk ( analog of muc2 with thr changed to ser)cht 24appahgvstapdtrpapgc ( analog of muc1a with an interchange of thr and ser ) 
 for clarity in the synthetic peptides cht 1  cht 22 the thr residue is bold and the amino acid residues which are varied are underlined acceptor peptides used in this study 
 for clarity in the synthetic peptides cht 1  cht 22 the thr residue is bold and the amino acid residues which are varied are underlined the enzyme , ppgalnact , without cytoplasmic tail and transmembrane domain ( amino acid 126 ) was expressed and purified exactly as in . a further truncated version , ppgalnact , omitting the complete lectin domain ( lacking amino acid 477600 ) was obtained in the same way . protein concentrations were determined by the micro - bca protein assay ( pierce , bonn , germany ) with bovine serum albumin as the standard . the enzyme activity of ppgalnact and ppgalnact was determined in 20  l reaction mixture containing 50  mm mes ( 2-(n - morpholino)ethanesulfonic acid ) , ph 7.0 , 10  mm mncl2 , 2  mm udp - galnac ( sigma - aldrich , vienna , austria ) , 10  nmol acceptor peptide ( cellmano biotech co. , ltd . , shanghai , china ) and 2  l enzyme solution ( ppgalnact or ppgalnact , protein concentrations 5.0  g / ml ) at 37  c for 90  min . for determination of donor specificity udp - gal , udp - glcnac , gdp - fucose , udp - xylose , udp - glucuronic acid ( all from sigma - aldrich , vienna , austria ) and cmp - neuraminic acid ( jennewein biotechnologie gmbh , germany ) the standard assay was conducted for 30  h and aliquots were taken at 1 , 2 , 4 , 6 , 8 , 23 , 26 and 30  h. for determination of the position of the transferred galnac - residues within the peptide , incubation was carried out with muc1a , muc1a , muc5ac and muc2 , respectively ( for sequences see table  1 ) , for 24  h analogously to the standard assay with additions of 10  nmol udp - galnac and 2  l of enzyme solution after 4 , 8 and 20  h. each assay was carried out at least in duplicate with appropriate controls . quantitative analysis was performed by hplc on a reversed phase c18 column ( 4.6    250  mm , 5  m , thermo scientific , vienna , austria ) in 0.5  % trifluoroacetic acid in water , applying a linear gradient from 15 to 25  % of eluent ( 0,1  % trifluoroacetic acid in acetonitrile ) in 20  min at a flow rate of 1  ml / min with a detection at 220  nm . mono - glycosylated muc2 and muc5ac were obtained using the standard assay conditions with 2  h of incubation time , followed by preparative runs on hplc exactly as for the quantification analysis . maldi - tof ms analysis was carried out on an autoflex speed maldi - tof ( bruker daltonics , germany ) equipped with a 1000  hz smartbeam.ii laser in positive mode using -cyano-4-hydroxycinnamic acid as matrix ( 1  % in 65  % acetonitrile solution ) . for crystallization 1  l of an 1:40 dilution of the samples was spotted on the plate , air dried , covered by 1  l of matrix solution and again air dried . ppgalnact activity assay solutions were purified via c-18 spe cartridges ( 25  mg , thermo scientific ) . briefly , the cartridges were equilibrated with 500  l methanol , 500  l 65  % acetonitrile and washed twice with 500  l 0,1  % formic acid . sample was applied , washed twice with 500  l 0,1  % formic acid and muc peptide was eluted with 65  % acetonitrile . for direct infusion etd - ms , the samples were vacuum - dried and redissolved in 50  % acetonitrile . muc peptides were directly infused into a bruker amazon speed etd ion trap using a hamilton syringe at a flow rate of 2  l / min . the mass spectrometer was operated in manual ms(n ) mode with etd as fragmentation mode . triply or four times charged precursors ions were measured with the following settings : icc target 200000 , maximum accumulation time 10  ms , isolation width 4  amu , etd reagent time 60100  ms . data were recorded for about 1015  min for every glycopeptide form and analysed with bruker s data analysis 4.0 . average mass spectra were generated using the snap peak finder algorithm and exported to bruker s biotools 3.2 . via the sequence editor , muc peptide sequences with additional galnac modification(s ) on every possible site ( ser or thr ) were generated and sent to biotools . theoretical peptide fragment masses ( c- and z - type ions ) were calculated for every possible glycopeptide form and compared to measured fragment masses . the identification of fragment ions , specific for a certain galnac modification site enabled us to illuminate the different glycosylation sites on the different muc peptide species [ 15 , 16 ] . the donor specificity of snail ppgalnact was investigated using standard assay conditions with the acceptor substrates muc2 and muc5ac peptide as well as the corresponding mono - glycosylated peptides and the nucleotide sugars udp - gal , udp - glcnac , gdp - fucose , udp - xylose , udp - glucuronic acid and cmp - neuraminic acid instead of udp - galnac . in none of these experiments any addition of a sugar was observed by maldi ( data not shown ) . obviously , the snail enzyme is strictly specific to udp - galnac . in contrast , the human ppgalnact2 has been found to utilize also udp - gal . previously we have shown by maldi - tof analysis that the snail ppgalnact transfers up to eight galnac residues to muc2 peptide . in order to obtain more information about the sites and a possible order of glycosylation , we analysed a large scale incubation of muc2 with esi - etd - ms / ms . muc2 forms with 18 galnac residues were selected as precursors for the etd reaction and analysed consecutively . due to the heterogeneity of the higher glycosylated muc2 forms ( especially the one with 5 galnacs ) , conclusion only about the major glycoform nevertheless , an order of glycosylation of the different sites of modification by ppgalnact could be deduced . thr-15 was clearly identified as the transferase s first choice . with three pro residues at positions 1 , + 1 and + 3 , this glycosylation site displays the previously published prerequisites for an ideal o - glycosylation site   (  +  or refers to residues that are n - terminal or c - terminal to a hydroxyl amino acid . for thr-2 they are in position 1 and + 3 , whereas for thr-13 pro is located at + 1 and + 3 . thr-17 with pro in 1 and + 1 is the subsequent glycosylation site , almost equal to thr-10 , which has no pro anywhere close . thr-19 , with two pro at 1 and 3 , but none on the other side , is next target , followed by thr-8 and thr-4 ( table  2 , fig .  1 ) . glycosylation at thr-21 was detected in a low amount and only in the form with 8 galnac residues , and only when no galnac was present linked to thr-19 . thus , even with 14 thr residues present in muc2 , only 9 potential glycosylation sites were identified , of which a maximum of 8 were in use , since thr-19 and thr-21 were never glycosylated in the same peptide ( fig .  1 ) . table 2ranking of thr residues in muc2 due to their glycosylation feasibilityrankthr residue1t-152t-2 , t-133t-10 , t-174t-195t-86t-47t-21fig . muc2 peptide with eight galnac residues [ m+4h ] at 1018  m / z was taken as a precursor for the etd reaction . two different forms ( indicated with  and * ) , slightly differing in modification site of the most c - terminal galnac were identified ( see insert ) . fragment ions marked with  or * enabled the differentiation between the two glycopeptide forms ranking of thr residues in muc2 due to their glycosylation feasibility annotated etd - ms / ms spectrum . muc2 peptide with eight galnac residues [ m+4h ] at 1018  m / z was taken as a precursor for the etd reaction . two different forms ( indicated with  and * ) , slightly differing in modification site of the most c - terminal galnac were identified ( see insert ) . fragment ions marked with  or * enabled the differentiation between the two glycopeptide forms also in muc5ac , muc1a and muc1a ( which is a shorter version of muc1a ) the glycosylation sites where determined . in the sequence of muc5ac ser-5 was the preferred acceptor site probably due to surrounding pro residues at position 1 , + 1 and + 3 . with longer incubation times up to four galnac residues could be attached . for muc1 and muc1a only the thr residues were identified as possible glycosylation sites ( thr-8 and thr-5 respectively ) . after an interchange of thr and ser in the sequence of muc1a ( thr-8 ser-8 and ser-9 thr-9 ) , the peptide ( cht 24 ) was no longer an acceptor for snail ppgalnact . cht 23 , an analogue of muc2 with all thr residues changed to ser residues , had a drastically reduced galnac transfer rate . while on muc2 peptide 8 galnac residues could easily be attached , only four galnac residues could be transferred to cht 23 , even after the longest incubation time . in order to investigate the optimal amino acid sequence close to a potential o - glycosylation site , 22 synthetic variants of a good acceptor peptide with one single thr and no ser were tested ( table  1 ) . the model acceptor peptide was designed based on earlier studies of human ppgalnact2 [ 1821 ] . our variation study included only three amino acids upstream and downstream from the glycosylation site , still the length of the chosen peptide was 17 amino acids in total , in order to avoid activity reductions due to too small chains . about 30  % of muc1a peptide ( 19 amino acids ) was converted into galnac - muc1a in the standard incubation assay , whereas muc1a peptide , a shorter version of muc1a with 11 amino acids , was converted to only 6  % ( fig . 2incorporation rate of galnac residues into native acceptor peptides incorporation rate of galnac residues into native acceptor peptides the central amino acid of the model peptide was a thr surrounded by four pro residues in position 3 , 1 , + 1 and + 3 and two gly at 2 and + 2 . these pro and gly residues were changed , while the five amino acids at the n- and at the c - terminus , respectively , were kept the same for all peptides . glycosylated products of the standard incubation assay were analysed by maldi - tof ms and quantified by hplc . the acceptor peptides were sorted into 4 groups according to acceptor quality ( table  3 ) : group 1  complete glycosylation within the time of the standard assay ; group 2  over 50  % ; group 3 549  % ; and group 4 - only traces were glycosylated . for those peptides which were completely converted within the standard conditions ( cht 1 , cht 3 , cht 9 , cht 10 , cht 11 , cht 12 , cht 13 ) , this revealed that for three of them ( cht 3 , cht 9 , cht 12 ) the change of one pro ( position 1 : prohis ; + 1 : proglu ; + 3 : protyr ) enhanced the quality of the acceptor , all the others slightly reduced incorporation compared with cht 1 . changing the gly in position + 2 to arg ( cht 11 ) did not affect the incorporation while the change to another basic amino acid ( his ) led to a drastic decrease ( cht 5 ) . only a moderate decrease was seen changing at 2 glyval ( cht 13 ) or at 1 proval ( cht 2 ) . combining all changes with moderate effect ( his - val - val - thr - glu - arg - tyr , cht 15 ) , activity was completely abolished.table 3peptides listed by their quality as acceptors for snail ppgalnact . for clarity only those amino acids group 1 : very good acceptors ( 95100% of acceptor conversion using standard conditions ) , group 2 moderate acceptors ( 5094% conversion ) , group 3 : weak acceptors ( 549% conversion ) , group 4 : incorporation less than 5% 
 ideal acceptor peptide for vertebrate ppgalnact2 according to   peptides listed by their quality as acceptors for snail ppgalnact . for clarity group 1 : very good acceptors ( 95100% of acceptor conversion using standard conditions ) , group 2 moderate acceptors ( 5094% conversion ) , group 3 : weak acceptors ( 549% conversion ) , group 4 : incorporation less than 5% 
 ideal acceptor peptide for vertebrate ppgalnact2 according to   a similar example for a poor combination is cht 6 . changing both pro to tyr at + 3 , which alone increased activity ( cht 3 ) , and pro to val at 1 , which alone only moderately decreased activity ( cht 2 ) resulted in a significant decrease of acceptor quality ( cht 6 ) . but also an increase of activity could be observed by combining some changes that decreased acceptor activity previously : a change of glyala at + 2 gives a rather weak acceptor ( cht 22 ) . but this change together with glyphe at 2 and proval at 1 ( cht 18 ) increased acceptor quality . if there was an additional change of proasp at 3 a further activity increase was detected ( cht 16 ) while proval or ile at 3 had a less pronounced effect ( cht 20 , cht 19 ) , yet , was still better than ala alone ( cht 22 ) . when the proasp change at 3 was combined with ala at + 2 and phe at 2 or val at 1 ( cht 17 , cht 21 ) , the activity was less than for ala alone ( cht 22 ) . the 1 position was found to be especially sensitive to changes . there , proval yielded in moderate reduction ( cht 2 ) while proile , leu or phe resulted in a strong decrease ( cht 7 , cht 14 ) or complete abolishment of activity ( cht 8) . however , the change of pro to ile at + 1 resulted only in a slightly weaker acceptor quality ( cht 10 ) . taken together , the results obtained with the synthetic peptides , pro residues in 3 , 1 , + 1 and + 3 favoured galnac addition , but other amino acids may be also present ( 3 : his , asp ; 2 : val , phe ; 1 : val ; + 1 : glu , ile ; + 2 : arg , ala , his ; + 3 : tyr ) . there seems to be no strict requirement for a certain amino acid at a specific position except that some amino acids completely prevented transfer ( 1 : ile , leu , phe ; + 3 : lys ) . generally , we found that the more amino acids we changed in the acceptor peptide that were previously identified as  ideal  for vertebrates , the less efficient the acceptor became . etd - ms / ms , which had been already applied by yoshimura   to identify the glycosylation sequence carried out by human ppgalnact3 on muc5ac , was used for revealing the incorporation of galnac into the native acceptors muc1a , muc1a , muc2 and muc5ac . thr-8 of muc1a is flanked by the sequence his - gly - val upstream and ser - ala - pro downstream , which is for sure a good variant . in contrast , thr-13 is located between ala - pro - asp and arg - pro - ala , which is not surprisingly an unfavourable combination . shortening of the total chain ( muc1a ) did not change the preference but reduced the transfer rate . changing the thr to ser in the previously preferred glycosylation site of this peptide abolished its acceptor qualities totally ( cht 24 ) . this is surprising as a ser residue in a similar good neighbourhood thr - thr - pro - ser - pro - val - pro is the enzyme s first choice in muc5ac . thr-15 with pro in 1 , + 1 , and + 3 was the preferred acceptor site followed by thr residues with less pro residues in ideal positions ( see above ) . an already glycosylated thr in 2 position or in + 2 position seemed to enhance further glycosylation . a previous glycosylation of thr-19 prohibited any glycosylation at thr-21 as we never found glycosylated products on both of these sites . a big exception to all these empirical rules was the fast glycosylation of thr-10 despite the lack of pro in close neighbourhood . there is a val at + 2 and an already glycosylated thr at + 3 ; the rest are not glycosylated thr residues . the already glycosylated thr may be the reason of the conversion of the sequence into an attractive acceptor . all native and synthetic peptides were also tested using an enzyme variant devoid of the lectin domain ( ppgalnact ) . no differences were observed for group 1 and group 4 . in group 2 and 3 a change in 2 position had more influence on the activity towards an acceptor without lectin domain than on the complete enzyme . cht 13 with glyval in position 2 turned out to be a weaker acceptor for ppgalnact . only 46  % incorporation was detected by the enzyme without lectin domain compared to 100  % transfer by the enzyme containing the lectin domain . while phe at that position often slightly increased the transfer ( cht 16 : 7080  % , cht 18 : 7075  % ) . for the native substrates muc2 and muc5ac we performed a time course with the complete enzyme and with ppgalnact . samples were analysed by hplc and maldi - tof ms after 1 , 2 , 4 , 6 , 8 , 23 , 26 and 30  h. both enzyme variants incorporated the same number of galnac residues ( maximum 8 in muc2 and 4 in muc5ac ) and exhibited the same incorporation rate . remarkably , there was no statistical significant difference in the transfer rate comparing the enzyme variants with and without lectin domain ( fig . 3).fig . 3maldi - tof ( [ m+ na ] ) analysis of the transfer of galnac residues using muc5ac , muc2 or cht 23 as acceptor peptide . acceptor peptide without incubation ( a , d , g ) ; 8-h - incubation with ppgalnact containing the lectin domain ( b , e , h ) ; 8-h - incubation with ppgalnac without the lectin domain ( c , f , i ) . the numbers indicate the incorporated galnac residues maldi - tof ( [ m+ na ] ) analysis of the transfer of galnac residues using muc5ac , muc2 or cht 23 as acceptor peptide . acceptor peptide without incubation ( a , d , g ) ; 8-h - incubation with ppgalnact containing the lectin domain ( b , e , h ) ; 8-h - incubation with ppgalnac without the lectin domain ( c , f , i ) . in order to investigate the optimal amino acid sequence close to a potential o - glycosylation site , 22 synthetic variants of a good acceptor peptide with one single thr and no ser were tested ( table  1 ) . the model acceptor peptide was designed based on earlier studies of human ppgalnact2 [ 1821 ] . our variation study included only three amino acids upstream and downstream from the glycosylation site , still the length of the chosen peptide was 17 amino acids in total , in order to avoid activity reductions due to too small chains . about 30  % of muc1a peptide ( 19 amino acids ) was converted into galnac - muc1a in the standard incubation assay , whereas muc1a peptide , a shorter version of muc1a with 11 amino acids , was converted to only 6  % ( fig . . 2incorporation rate of galnac residues into native acceptor peptides incorporation rate of galnac residues into native acceptor peptides the central amino acid of the model peptide was a thr surrounded by four pro residues in position 3 , 1 , + 1 and + 3 and two gly at 2 and + 2 . these pro and gly residues were changed , while the five amino acids at the n- and at the c - terminus , respectively , were kept the same for all peptides . glycosylated products of the standard incubation assay were analysed by maldi - tof ms and quantified by hplc . the acceptor peptides were sorted into 4 groups according to acceptor quality ( table  3 ) : group 1  complete glycosylation within the time of the standard assay ; group 2  over 50  % ; group 3 549  % ; and group 4 - only traces were glycosylated . for those peptides which were completely converted within the standard conditions ( cht 1 , cht 3 , cht 9 , cht 10 , cht 11 , cht 12 , cht 13 ) , the experiment was repeated with a quarter of the previous amount of enzyme . this revealed that for three of them ( cht 3 , cht 9 , cht 12 ) the change of one pro ( position 1 : prohis ; + 1 : proglu ; + 3 : protyr ) enhanced the quality of the acceptor , all the others slightly reduced incorporation compared with cht 1 . changing the gly in position + 2 to arg ( cht 11 ) did not affect the incorporation while the change to another basic amino acid ( his ) led to a drastic decrease ( cht 5 ) . only a moderate decrease was seen changing at 2 glyval ( cht 13 ) or at 1 proval ( cht 2 ) . combining all changes with moderate effect ( his - val - val - thr - glu - arg - tyr , cht 15 ) , activity was completely abolished.table 3peptides listed by their quality as acceptors for snail ppgalnact . for clarity only those amino acids group 1 : very good acceptors ( 95100% of acceptor conversion using standard conditions ) , group 2 moderate acceptors ( 5094% conversion ) , group 3 : weak acceptors ( 549% conversion ) , group 4 : incorporation less than 5% 
 ideal acceptor peptide for vertebrate ppgalnact2 according to   peptides listed by their quality as acceptors for snail ppgalnact . for clarity only those amino acids are given that are changed compared to the model peptide . group 1 : very good acceptors ( 95100% of acceptor conversion using standard conditions ) , group 2 moderate acceptors ( 5094% conversion ) , group 3 : weak acceptors ( 549% conversion ) , group 4 : incorporation less than 5% 
 ideal acceptor peptide for vertebrate ppgalnact2 according to   a similar example for a poor combination is cht 6 . changing both pro to tyr at + 3 , which alone increased activity ( cht 3 ) , and pro to val at 1 , which alone only moderately decreased activity ( cht 2 ) resulted in a significant decrease of acceptor quality ( cht 6 ) . but also an increase of activity could be observed by combining some changes that decreased acceptor activity previously : a change of glyala at + 2 gives a rather weak acceptor ( cht 22 ) . but this change together with glyphe at 2 and proval at 1 ( cht 18 ) increased acceptor quality . if there was an additional change of proasp at 3 a further activity increase was detected ( cht 16 ) while proval or ile at 3 had a less pronounced effect ( cht 20 , cht 19 ) , yet , was still better than ala alone ( cht 22 ) . when the proasp change at 3 was combined with ala at + 2 and phe at 2 or val at 1 ( cht 17 , cht 21 ) , there , proval yielded in moderate reduction ( cht 2 ) while proile , leu or phe resulted in a strong decrease ( cht 7 , cht 14 ) or complete abolishment of activity ( cht 8) . however , the change of pro to ile at + 1 resulted only in a slightly weaker acceptor quality ( cht 10 ) . taken together , the results obtained with the synthetic peptides , pro residues in 3 , 1 , + 1 and + 3 favoured galnac addition , but other amino acids may be also present ( 3 : his , asp ; 2 : val , phe ; 1 : val ; + 1 : glu , ile ; + 2 : arg , ala , his ; + 3 : tyr ) . there seems to be no strict requirement for a certain amino acid at a specific position except that some amino acids completely prevented transfer ( 1 : ile , leu , phe ; + 3 : lys ) . generally , we found that the more amino acids we changed in the acceptor peptide that were previously identified as  ideal  for vertebrates , the less efficient the acceptor became . etd - ms / ms , which had been already applied by yoshimura   to identify the glycosylation sequence carried out by human ppgalnact3 on muc5ac , was used for revealing the incorporation of galnac into the native acceptors muc1a , muc1a , muc2 and muc5ac . thr-8 of muc1a is flanked by the sequence his - gly - val upstream and ser - ala - pro downstream , which is for sure a good variant . in contrast , thr-13 is located between ala - pro - asp and arg - pro - ala , which is not surprisingly an unfavourable combination . shortening of the total chain ( muc1a ) did not change the preference but reduced the transfer rate . changing the thr to ser in the previously preferred glycosylation site of this peptide abolished its acceptor qualities totally ( cht 24 ) . this is surprising as a ser residue in a similar good neighbourhood thr - thr - pro - ser - pro - val - pro is the enzyme s first choice in muc5ac . thr-15 with pro in 1 , + 1 , and + 3 was the preferred acceptor site followed by thr residues with less pro residues in ideal positions ( see above ) . an already glycosylated thr in 2 position or in + 2 position seemed to enhance further glycosylation . a previous glycosylation of thr-19 prohibited any glycosylation at thr-21 as we never found glycosylated products on both of these sites . a big exception to all these empirical rules was the fast glycosylation of thr-10 despite the lack of pro in close neighbourhood . there is a val at + 2 and an already glycosylated thr at + 3 ; the rest are not glycosylated thr residues . the already glycosylated thr may be the reason of the conversion of the sequence into an attractive acceptor . all native and synthetic peptides were also tested using an enzyme variant devoid of the lectin domain ( ppgalnact ) . no differences were observed for group 1 and group 4 . in group 2 and 3 a change in 2 position had more influence on the activity towards an acceptor without lectin domain than on the complete enzyme . cht 13 with glyval in position 2 turned out to be a weaker acceptor for ppgalnact . only 46  % incorporation was detected by the enzyme without lectin domain compared to 100  % transfer by the enzyme containing the lectin domain . while phe at that position often slightly increased the transfer ( cht 16 : 7080  % , cht 18 : 7075  % ) . for the native substrates muc2 and muc5ac we performed a time course with the complete enzyme and with ppgalnact . samples were analysed by hplc and maldi - tof ms after 1 , 2 , 4 , 6 , 8 , 23 , 26 and 30  h. both enzyme variants incorporated the same number of galnac residues ( maximum 8 in muc2 and 4 in muc5ac ) and exhibited the same incorporation rate . remarkably , there was no statistical significant difference in the transfer rate comparing the enzyme variants with and without lectin domain ( fig .  3).fig . 3maldi - tof ( [ m+ na ] ) analysis of the transfer of galnac residues using muc5ac , muc2 or cht 23 as acceptor peptide . acceptor peptide without incubation ( a , d , g ) ; 8-h - incubation with ppgalnact containing the lectin domain ( b , e , h ) ; 8-h - incubation with ppgalnac without the lectin domain ( c , f , i ) . the numbers indicate the incorporated galnac residues maldi - tof ( [ m+ na ] ) analysis of the transfer of galnac residues using muc5ac , muc2 or cht 23 as acceptor peptide . acceptor peptide without incubation ( a , d , g ) ; 8-h - incubation with ppgalnact containing the lectin domain ( b , e , h ) ; 8-h - incubation with ppgalnac without the lectin domain ( c , f , i ) . despite a considerable phylogenetic distance , the recently identified ppgalnact from the snail biomphalaria glabrata displays homology to human ppgalnact , especially to ppgalnact2 ( 74  % positives with 58  % identity according to ncbi blast analysis ) . here , we aimed at a characterization of the donor and acceptor substrate specificity to find out about parallels or differences between snail ppgalnact and human ppgalnact2 , which is the only crystallised   and the best characterised member of the ppgalnact2 homologs . the core regions of our synthetic peptides used in this study were variants of pro - gly - pro - thr - pro - gly - pro , which had been identified by gerken et al . to be an ideal acceptor peptide for human ppgalnact2 . in order to overcome the activity reduction seen with too small chains we added 5 amino acids on both ends of this core , resulting in peptides containing 17 amino acids . in previous studies on vertebrate ppgalnact2 and on the drosophila ortholog pgant2 the enhancing activity of pro residues close to the glycosylation site had been determined [ 19 , 2325 ] . especially the 1 position was found to be particularly relevant . charged amino acids in general decreased the activity , while ser , thr , ala and gly residues seemed to be acceptable in most of the positions . large , especially aromatic side chains abolished activity [ 19 , 20 , 24 , 26 ] . on the basis of the evaluation of a large set of o - glycosylation sites of vertebrate proteins , elhammer et al . developed an algorithm for the prediction of probable glycosylation sites . the accuracy of the method is about 7585  % [ 19 , 27 ] . in general , the results found for vertebrate ppgalnacts correlate with the data we received for the biomphalaria glabrata enzyme [ 2831 ] . ala had been identified as the most favourable amino acids for the human ppgalnact2 and an in vivo system using cos7 cells [ 18 , 20 ] . the snail enzyme clearly preferred gly and arg but was less in favour for ala in this position . an enhancement by tyr at + 3 was found for human ppgalnact1 , but not for human ppgalnact2 or drosophila pgant2 [ 18 , 25 ] , albeit snail ppgalnact2 activity was elevated by tyr at + 3 . similar to human ppgalnact2 phe , ile , leu decreased activity significantly but val was not that bad for the snail enzyme as it had been described for the human one . similar to human ppgalnact2 pro at 1 and + 3 was very important , while glu at + 1 was less supportive for the human than for the snail enzyme . his at 3 prevented activity of human ppgalnact2 , however , the snail enzyme was enhanced by this modification . studying the order of o - glycosylation of muc5ac , we found that the snail ppgalnact2 used ser-5 rather than thr-9 as the first glycosylation site which is in contrast to human ppgalnact2 . this is surprising , as in all other cases the snail enzyme clearly preferred thr over ser residues , which is in accordance with other ppgalnact2s [ 17 , 19 ] . however , an early study on ppgalnacts using a preparation from bovine colostrum found that in vivo the preference may be different as compared to the in vitro requirements . as soon as the acceptors were already glycosylated , the situation became even more complex . while the above mentioned preferences for neighbouring amino acids were still visible , the glycosylation status of adjacent thr residues influenced the choice of attachment sites . we also did not observe a preference for c - terminally placed galnac - o - thr residues as has been shown for human ppgalnact2 . in agreement with earlier results from vertebrates [ 3336 ] , glycosylation of potential glycosylation sites next to an already occupied site was not detected . therefore , the snail enzyme is definitely not a  finishing  enzyme like human ppgalnact7 , ppgalnact10 or ppganact15 which add the final galnac residues to already highly glycosylated acceptors , even in neighbouring positions . thus , one may speculate that the snail genome harbours further ppgalnact homologs . in comparison with all other so far analysed ppgalnact2s from vertebrates and also from drosophila melanogaster , the enzyme from biomphalaria it has a broad specificity for non - glycosylated as well as glycosylated acceptor substrates . several studies have been performed discussing the influence of the lectin domain on the glycosylation behaviour . it was shown that the lectin domain was not necessary for catalytic activity of human ppgalnact2 but shifts in the preferred sites of glycosylation and higher densities of glycosylation may occur [ 3032 , 37 , 38 ] . for the snail enzyme we did not observe remarkable differences of transfer for acceptors with one single glycosylation site as well as for acceptors with multiple glycosylation sites , neither in speed of incorporation nor in the final number of attached galnac residues . taken together , this first thorough study of the substrate specificity of a ppgalnact from a snail reveals remarkable similarities to the transferases from vertebrates and insects . one can speculate that also molluscs contain more than one type of ppgalnact , like it has been shown for the other so far analysed species . considering several millions of years of separate evolution , this high degree of functional conservation strongly suggests a highly important common function or set of functions of protein o - glycosylation .
o - glycosylation is a widely occurring posttranslational modification of proteins . 
 the glycosylation status of a specific site may influence the location , activity and function of a protein . 
 the initiating enzyme of mucin - type o - glycosylation is udp - galnac : polypeptide galnac transferase ( ppgalnact ; ec 2.4.1.41 ) . using electron - transfer dissociation mass spectrometry , ppgalnact from the snail biomphalaria glabrata 
 was characterized regarding its ability to glycosylate threonine and serine residues in different peptide sequence environments . 
 the preferences of the snail enzyme for flanking amino acids of the potential glycosylation site were very similar to vertebrate and insect members of the family . 
 acceptor sites with adjacent proline residues were highly preferred , while other residues caused less pronounced effects . 
 no specific o - glycosylation consensus sequence was found . 
 the results obtained from synthetic peptides were in good correlation with the observed glycosylation patterns of native peptides and with the order of attachment in a multi - glycosylated peptide . 
 the snail enzyme clearly preferred threonine over serine in the in vitro assays . 
 no significant differences of transfer speed or efficiency could be detected using a mutant of the enzyme lacking the lectin domain . 
 this is the first characterisation of the substrate specificity of a member of the ppgalnact family from mollusc origin .