Proc. Natl. Acad. Sci. USAVol. 93, pp. 10111-10116, September 1996Biochemistry

a-Lactalbumin affects the acceptor specificity ofLymnaeastagnalis albumen gland UDP-GalNAc:GlcNAcf3-R131-*4-N-acetylgalactosaminyltransferase: Synthesisof GalNAc,81-4Glc

(snail/schistosome/lacdiNAc/complex-type glycans/glycosyltransferase)

ALEX P. NEELEMAN AND DIRK H. VAN DEN EIJNDEN*Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

Communicated by Stuart Kornfeld, Washington University School of Medicine, St. Louis, MO, June 12, 1996 (received for review March 2, 1996)

ABSTRACT The N,N'-diacetyllactosediamine (lacdiNAc)pathway of complex-type oligosaccharide synthesis is con-trolled by a UDP-GalNAc:GlcNAcf3-R j81-*4-N-acetylgalac-tosaminyltransferase (f34-GalNAcT) that acts analogously tothe common UDP-Gal:GlcNAcj8-R 131-*4-galactosyltrans-ferase ((34-GaiT). LacdiNAc-based chains particularly occurin invertebrates and cognate ,B4-GalNAcTs have been identi-fied in the snail Lymnaea stagnalis, in two schistosomal species,and in several lepidopteran insect cell lines. Because of thesimilarity in reactions catalyzed by both enzymes, we inves-tigated whetherL. stagnalis albumen gland (84-GalNAcT wouldshare with mammalian f34-GaIT the property of interactingwith a-lactalbumin (c-LA), a protein that only occurs in thelactating mammary gland, to form a complex in which thespecificity of the enzyme is changed. It was found that, underconditions where (84-GalT forms the lactose synthase complexwith a-LA, the snail .84-GalNAcT was induced by this proteinto act on Glc with a > 100-fold increased efficiency, resultingin the formation of the lactose analog GalNAc,81-4Glc. Thisforms the second example of a glycosyltransferase, the spec-ificity of which can be altered by a modifier protein. So far,however, no protein fraction could be isolated from L. stagnalisthat could likewise interact with the 134-GalNAcT. Neither hadlysozyme c, a protein that is homologous to a-LA, an effect onthe specificity of the enzyme. These results raise the questionof how the capability to interact with a-LA has been conservedin the snail enzyme during evolution without any apparentselective pressure. They also suggest that snail (4-GalNAcTand mammalian f84-GalT show similarity at a molecular leveland allows the identification of the f34-GalNAcT as a candi-date member of the f4-GaIT family.

In recent years an increasing number of animal glycoconju-gates have been described containing protein- or lipid-linkedcomplex-type glycans based on the disaccharideGalNAc,B1f34GlcNAc (N,N'-diacetyllactosediamine; lacdi-NAc) rather than on the common GalP31--4GlcNAc (N-acetyllactosamine; lacNAc) disaccharide unit (1). The group ofglycoconjugates that carry such chains is quite diverse andcomprises inter alia hormones, enzymes, membrane glycopro-teins, and transport proteins. It has been proposed thatlacdiNAc-based chains confer specific properties on the gly-coconjugates carrying them (2-5).LacdiNAc-based oligosaccharide chains appear to occur in

particular on glycoconjugates of invertebrates such as molluscs(6, 7), schistosomes (8, 9), nematodes (10, 11), and insects(12-14). In schistosomes (3, 15), the pond snail Lymnaeastagnalis (16), and several lepidopteran insect cell lines (17) a

UDP-GalNAc:GlcNAco3-R (1-*4-N-acetylgalactosaminyl-transferase (,34-GalNAcT) has been described that controlsthe synthesis of lacdiNAc units according to the followingreaction:

UDP-GalNAc + GlcNAcA,-R->

GalNAcf31 -4GlcNAcf3-R + UDP.

In mammals, a related enzyme has been described inpituitary gland that has been suggested to be glycoprotein-hormone specific in that it recognizes a Pro-Xaa-Arg/Lyssequence 6-9 amino acids upstream of the glycosylation site ofthese glycoproteins (18, 19). By contrast, the snail, schistoso-mal, and insect cell enzymes are polypeptide unspecific and acton any terminal 3-linked GlcNAc residue regardless of theunderlying structure (3, 15-17). In this respect the latter,34-GalNAcTs rather resemble the UDP-Gal:GlcNAcj-Rf1-4-galactosyltransferase (134-GalT) (EC 2.4.1.38) com-monly occurring in higher animal species where it controls thesynthesis of lacNAc-based complex-type glycans (20, 21).The 134-GalT has the unique property of being capable to

interact with a-lactalbumin (a-LA) to form the lactose-synthase complex (EC 2.4.1.22), in which the specificity of theenzyme is changed from catalyzing the transfer to terminalGlcNAc residues on glycoconjugates to transfer to free Glc,yielding lactose (20, 22-24). This typically happens duringlactation in the mammary gland of almost all mammals,including marsupials, upon the hormone-induced secretion ofa-LA by the glandular epithelial cells (20, 25-27). The thusroduced lactose is a major osmotic regulator in the process ofmilk secretion in addition to being the major carbohydratesource for the newborn (20, 27).

Because of the similarity of the reactions catalyzed bymammalian f34-GalT and the invertebrate f34-GalNAcT, andtheir overlapping acceptor specificities, we investigatedwhether the two enzymes would also share the capability tointeract with a--LA. Indeed, it was found that the snail ,B4-GalNAcT (but not the schistosomal enzyme) was responsive toa-LA and could be induced to catalyze the synthesis ofGalNAcfl3->4Glc in vitro by this modifier protein. It thusappears that the property of interacting with a-LA is shared bytwo different glycosyl transferases of evolutionary distantspecies.

Abbreviations: ag-GP-F2, degalactosylated asialo-glycopeptide fromhuman fibrinogen; (4-GalT, UDP-Gal:GlcNAcf3-R (1-*4-galactosyl-transferase; f34-GaINAcT, UDP-GalNAc:GlcNAcI3-R B1-4-N-acetylgalactosaminyltransferase; ,B4-GlcNAcT, UDP-GlcNAc:Glc-NAcI3-R 31-*4-N-acetylglucosaminyltransferase; a-LA, a-lactalbu-min; lacNAc (GalI134GlcNAc), N-acetyllactosamine; lacdiNAc(GalNAc/31-*4GlcNAc), N,N'-diacetyllactosediamine.*To whom reprint requests should be addressed. e-mail:DH.van den Eijnden.medchem@med.vu.nl.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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10112 Biochemistry: Neeleman and van den Eijnden

EXPERIMENTAL PROCEDURESMaterials. Bovine a-LA, lysozyme c (hen egg white), unla-

beled UDP-GalNAc and UDP-Gal were obtained from Sigma.UDP-hexanolamine-Sepharose 4B was prepared as described(28). UDP-[3H]GalNAc (8.7 Ci/mmol; 1 Ci = 37 GBq) andUDP-[3H]Gal (10.6 Ci/mmol) were purchased from NewEngland Nuclear. The nucleotide sugar donors were dilutedwith the unlabeled materials to give the desired specificradioactivity. All other chemicals were obtained from com-mercial sources and were of the best quality available. Glyco-peptide GP-F2 was prepared from desialylated human fibrin-ogen by pronase digestion as described (29). The peptideportion of GP-F2 consisted of over 90% of Gly-Glu-Asn andGlu-Asn in a ratio of 3:2. GP-F2 was enzymatically degalac-tosylated with jack bean 13-galactosidase (0.2 unit/,tmol ter-minal Gal) in 50 mM sodium acetate (pH 4.0) to yield theagalacto form of GP-F2 (ag-GP-F2, degalactosylated asialo-glycopeptide from human fibrinogen). Adult laboratory-bredspecimens of the pond snail L. stagnalis were obtained from theDepartment of Biology, Vrije Universiteit, Amsterdam. Al-bumen glands were dissected from the snails and stored at-20°C until use.Enzyme Preparations and Snail-Derived Protein Fractions.

,B4-GalNAcT from the albumen gland of L. stagnalis waspartially purified as described (16) and bovine colostrum134-GalT was purified according to Blanken et al. (30). Analbumen gland soluble fraction was obtained by high-speedcentrifugation (60 min at 100,000 x g) of a homogenate asdescribed (16). Snail plasma was prepared from the pooledhemolymph (blood) of 10 adult specimens by the addition of0.1 mM protease inhibitors (Sigma) and removal of thehemocytes by centrifugation (5 min at 100 x g).Assay of 184-GalNAcT and f34-GalT Activity. Standard in-

cubation mixtures contained 5 ,umol sodium cacodylate (pH7.0), 2.0 ,umol MnCl2, 0.25 ,tl Triton X-100, 12.5 nmol UDP-[3H]GalNAc (1.0 Ci/mol) or 12.5 nmol UDP-[3H]Gal (1.25Ci/mol), 90 nmol GlcNAc or 50 nmol ag-GP-F2 or 810 nmolGlc as acceptor and enzyme (3.8-6.9 ,ug protein, 10-16 ,tU)in a volume of 50 ,ul. Where indicated, a-LA (prepared as freshsolution), albumen gland soluble fraction, snail plasma, ly-sozyme c, and bovine serum albumin were added in theconcentrations indicated. The mixtures were incubated for30-60 min at 37°C. The reactions were terminated by theaddition of 0.5 ml cold H20. The samples were subsequentlypassed over Dowex 1-X8 (Cl- form) columns of 0.5 ml. Thecolumns were eluted twice with 0.5 ml H20, and the combinedeluates were counted for radioactivity after adding a liquidscintillation cocktail. Incorporation of radioactivity was cor-rected for the transfer obtained without exogenously addedacceptors. Kinetic parameters were estimated from Lineweav-er-Burk plots that were obtained by varying the GlcNAcconcentration from 0.2 to 50 mM and that of Glc from 1.0 to80 mM and from 10 mM to 1.6 M in the presence or absenceof 12 mg of a-LA per ml, respectively. In all assays the metalcofactor (Mn2+) was present at saturating (40 mM) conditions(16).

Large-Scale Incubation. A large-scale incubation with Glcas acceptor was conducted to isolate sufficient product forcharacterization by 400-MHz 1H-NMR spectroscopy. Theincubation mixture contained 130 ,umol sodium cacodylate(pH 6.5), 52 ,imol MnCl2, 6.5 ,lI Triton X-100, 7.9 ,umolUDP-[3H]GalNAc (12.5 mCi/mol), 780 ,tmol Glc, 0.02%sodium azide, 13 mg a-LA, and 0.429 mU of partially purifiedf4-GalNAcT in a volume of 1.3 ml. This mixture was incubatedfor 66 h at 37°C. Unused donor substrate (UDP-[3H]GalNAc)was removed by passing the mixture over a column (1.2 ml) ofDowex 1-X8 (Cl- form). The combined run-through and threewashes of 1 ml each were fractionated on a column (1.6 x 200cm) of Bio-Gel P-4 (200-400 mesh) equilibrated and eluted

with 0.05 M ammonium acetate (pH 5.2). Fractions wereassayed for radioactivity by liquid scintillation counting and forthe presence of free Glc by the phenol-sulfuric acid method(31). Fractions containing the radioactive product and no freeGlc were pooled and lyophilized.Product Identification. Methylation analysis was carried out

as described (32), except for the methylation step which wasperformed according to Ciucanu and Kerek (33). For theanalysis of the product by 400 MHz IH-NMR spectroscopy thematerial was desalted on a column (1.0 x 42 cm) of Bio-GelP-2 (200-400 mesh) run in water. The resulting product wastreated three times with 2H20 (99.75 atom %; Merck) at p2H7 and room temperature with intermediate lyophilization.Finally the sample was redissolved in 360 [lI of 2H20 (99.95atom %; Aldrich). 1H-NMR spectroscopy was performed at400 MHz on a Bruker MSL-400 spectrometer (Department ofPhysics, Vrije Universiteit, Amsterdam) operating in the Fou-rier-transform mode. The probe temperature was kept at 300K. Resolution enhancement of the spectra was achieved byLorentzian-to-Gaussian transformation. Chemical shifts areexpressed downfield from 4,4-dimethyl-4-silapentane-1-sulfonate, but were actually measured by reference to internalacetone (8 = 2.225 ppm).

RESULTSEffect of a-LA on the Acceptor Specificity of J34-GalNAcT.

The effect of a-LA on the activity of snail albumen gland,B4-GalNAcT with GlcNAc and Glc as acceptors is shown inFig. 1. In the absence of a-LA, the activity with GlcNAc (at aconcentration of 1.8 mM) was 21.0 mU.mg-1 protein, whereasthe activity with Glc (16.2 mM) was only 3.1 mU-mg-1 protein.The enzyme activity with GlcNAc increased with increasingconcentrations of a-LA. No inhibition was observed at anyconcentration tested. At the highest concentration employed(12 mgml-1) the activity with GlcNAc was almost 3-foldhigher as compared with that without a-LA. When GlcNAcwas present at a concentration of 10 mM, the enzyme activitywas enhanced about 2-fold at 12 mg-ml-' a-LA. The activityof the f4-GalNAcT with Glc was strongly stimulated by a-LAin a concentration-dependent manner. At the highest a-LA

300-

*200

(0EN

aI)a) 1009.>/IO

0 1 2 3 4 5 12

[a-Lactalbumin] Cmg/mi)

FIG. 1. Effect of the concentration of a-LA on the incorporationof GalNAc from UDP-GalNAc catalyzed by L. stagnalis albumengland ,34-GalNAcT into GlcNAc (-) and Glc (0), respectively, usingthe standard incubation conditions described under ExperimentalProcedures.

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Table 1. Comparison of the substrate specificities of snail albumengland 034-GalNAcT and bovine colostrum ,B4-GalT

Absence ~ or Relative activity, %Donor Acceptor presence (+) of Snail Bovine

substrate substrate a-LA ,B4-GalNAcT f34-GalTUDP-GalNAc GlcNAc - 100 0.3UDP-GalNAc GlcNAc + 279 5.0UDP-GalNAc ag-GP-F2 - 363 1.3UDP-GalNAc ag-GP-F2 + 318

10114 Biochemistry: Neeleman and van den Eijnden

H-1GaINAc8 NAc

GaINAc5

rIujlcp H-2

H-iGIca V Ic

I ,[~~~~~~~I; 1I ~~~~~-x1

II/,nI,i,

5.2 4.8 4.4 4.0 3.6 3.2 2.86 (ppm)

2.2 1.8

FIG. 2. Diagnostic areas of the 400 MHz 1H-NMR spectrum of thedisaccharide product (GalNAc01j3l4Glc) obtained by incubatingUDP-GalNAc and Glc with albumen gland ,B4-GalNAcT in thepresence of a-LA.

the H-1 signals of Gal in GalBl-14Glc compared withGal,31->4GlcNAc. Similarly, the H-1 resonances of the reduc-ing sugar in the product have positions relative to those of Glcin Gal,l-3>4Glc as have the H-1 resonances of GlcNAc inGalNAcJ31-4GlcNAc relative to those of Gall1- >4GlcNAc.Finally, a double doublet characteristic of the H-2 of a Glcresidue (P3-anomer) was found in the spectrum of the product.These results along with those of the methylation analysis andthe linkage specificity (,B1-*4) of the f34-GalNAcT (16) showthat the structure of the product formed was GalNAc3l-*>Glc.

Effect of Proteins and Snail-Derived Protein Fractions on,34-GalNAcT Activity. To investigate whether lysozyme, whichis structurally and evolutionary related to a-LA (20, 38, 39),would also be capable to modify the specificity of snail,B4-GalNAcT and whether L. stagnalis would produce proteinswhich also could interact with the enzyme, several proteins andsnail-derived fractions were tested for their capability toinduce the enzyme to synthesize GalNAc,13->4Glc. Apartfrom a-LA, none of these proteins and fractions tested showedany effect on the activity of the 134-GalNAcT with Glc as anacceptor (Table 5). Also, these proteins and fractions had nomajor effect on the activity of the ,34-GalNAcT with GlcNAcas an acceptor except for the albumen gland supernatant. Thestimulatory effect of this supernatant (+73%) on the enzymeactivity with the latter substrate could be accounted for by the,B4-GalNAcT activity contained in this fraction.

DISCUSSIONAn early milestone in the study of glycosyltransferases was thefinding reported almost 30 years ago that the specificity of

Table 5. Effect of proteins and snail-derived protein fractions onthe activity of albumen gland ,B4-GalNAcT with GlcNAc (1.8 mM)and Glc (16.2 mM) as acceptors, and UDP-GaINAc as donor substrate

Activity, %

Addition GlcNAc Glc

None 100 3.6a-LA 298 153Lysozyme c 39 1.8Bovine serum albumin 84 5.2Albumen gland soluble

fraction 173 4.9Snail plasma 99 3.3

One hundred percent corresponds to an activity of 17.6 mU mg-1protein. a-LA, lysozyme, bovine serum albumin, albumen glandsoluble fraction, and snail plasma were present at a final concentrationof 12, 12, 12, 3.8 and 0.41 mg protein-ml-1, respectively.

,B4-GaiT is modified from acting on GlcNAc,3-R acceptors toacting on Glc by the milk protein a-LA (22, 23). Only recentlyit has been reported that a-LA in addition induces f4-GalT toutilize UDP-GalNAc rather than UDP-Gal in its action on freeGlcNAc (35). So far no other examples are known of glyco-syltransferases that show an altered specificity in the presenceof a modifier molecule. Our study shows, however, that a-LAnot only affects the specificity of ,B4-GalT, but also that of the34-GalNAcT from L. stagnalis albumen gland, a snail female

accessory sex gland that secretes the perivitelline fluid aroundthe fertilized eggs (40). Therefore this f34-GalNAcT forms thesecond example of a glycosyltransferase, the acceptor speci-ficity of which can be modified by another protein.The striking similarity in acceptor substrate requirements

(16, 20, 21) along with the shared responsiveness of the34-GalT and snail ,34-GalNAcT to a-LA suggests a relation-

ship at a molecular level (see ref. 41). Apart from the fact thatthese enzymes occur in two evolutionary distant species, sucha relationship would show a remarkable analogy with thatdescribed for the polymorphic blood group A and B trans-ferases. These enzymes likewise have the same acceptorspecificity and differ in the nucleotide sugar that is utilized,UDP-GalNAc and UDP-Gal, respectively (42). At a molecularlevel the cDNAs of the blood group enzymes differ only inseven bases, resulting in four amino acid differences in thepolypeptides (42, 43). Prompted by this analogy we attemptedto clone the cDNA of the ,B4-GalNAcT from snail libraries byheterologous hybridization using a ,B4-GalT cDNA as a probe(44). Interestingly, this has led to the cloning of a relatedUDP-GlcNAc:GlcNAcf3-R ,1-->4-N-acetylglucosaminyltrans-ferase (,34-GlcNAcT) (but so far not yet to a ,34-GalNAcT)with specificity for acceptors with a terminal, 3-linked GlcNAcresidue, the existence of which was not predicted from oligo-

Table 4. NMR data of the product (GalNAcIl1-*4Glc) formed by the action of albumen gland ,B4-GalNAcT on Glc and comparison with thedata of three related disaccharides

Anomer Chemical shift (coupling constant), ppm (Hz)Reporter ofgroup Residue compound Product Gal,B1-*4Glc Galp3l-*4GlcNAc GalNAc,B1 >4GlcNAc*

H-1 Glc a 5.205 (3.8) 5.223 (3.7)13 4.650 (8.0) 4.664 (7.9)

H-2 Glc 13 3.263 (9.3) 3.286 (8.8)H-1 GlcNAc a 5.206 (2.7) 5.194 (2.8)

13 4.725 (7.6) 4.702 (8.3)H-1 Gal a/03 4.451 (7.8) 4.478 (7.8)H-1 GalNAc a 4.511 (8.5) 4.532 (8.4)

a 4.503 (8.4) 4.522 (8.4)NAc-CH3 GlcNAc a/f3 2.043 2.037

GalNAc a/f3 2.063 2.067Note that coupling constants represent J1,2 values except for H-2 of Glc (J2,3)-

*Data taken from ref. 37.

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saccharide-structural data (44). It has been proposed that the,B4-GlcNAcT along with the f34-GalTs and the invertebrate,B4-GalNAcTs constitute a family of enzymes (45) which haveevolved from a common ancestor (H. Bakker, T. Sato, A. VanTetering, K. Furukawa, I. van Die and D.H.v.d.E., unpublisheddata). The enzymes within this family have in common thatthey catalyze the transfer of Gal, GalNAc, or GlcNAc, fromthe respective UDP-sugar donors, in 61-4-linkage to a ter-minal I3-GlcNAc in an acceptor. Thereby they would eachcontrol a separate route of complex-type oligosaccharidebiosynthesis, the lacNAc-, lacdiNAc- and chitobio-pathway,respectively (45).Although P4-GalT and P4-GalNAcT are similarly respon-

sive to a-LA in that Glc is utilized at a two to three orders ofmagnitude higher kinetic efficiency in the presence of thisprotein (refs. 20 and 34 and this study), there is a noticeabledifference in the effect observed with GlcNAc. While theactivities of bovine milk (refs. 23 and 34 and this study),tammar wallaby mammary gland (25) and chicken serum (46)f34-GalTs with this acceptor (at concentrations >2-3 mM) arestrongly reduced in the presence of a-LA, the activity of snail134-GalNAcT with GlcNAc is markedly elevated by this pro-tein, also at higher concentrations of the acceptor. In addition,it should be noted that not all f34-GalTs and 134-GalNAcTs areresponsive to a-LA. In porcine trachea a j4-GalT has beendescribed that is insensitive to this modifier protein (47) anda ,B4-GalT with the same property was found in the adultworms of Schistosoma mansoni (48). Interestingly, a ,B4-GalNAcT recently described in cercariae of the avian schis-tosome Trichobilharzia ocellata (3), which shows an acceptorspecificity that is very similar to that of the snail f34-GalNAcT(16), also appeared to be nonresponsive to a-LA (data notshown). Finally, the specificity of the snail f34-GlcNAcT men-tioned above was not modified by this protein either (H.Bakker, P. S. Schoenmakers, D. H. Joziasse, I. Van Die, andD.H.v.d.E., unpublished data). It thus appears that some, butnot all members of the p4-GalT/134-GalNAcT/134-GlcNAcTfamily (45) can interact with a-LA.

Studies to define the regions of 834-GalT involved in itsinteraction with a-LA have been carried out with monoclonalantibodies (49) and specific amino acid modifying reagents(50). These have revealed that a-LA interacts with the NH2terminal domain of ,B4-GalT (150 amino acid residues in thesoluble form of the enzyme) comprising the stem region inaddition to (part of) the acceptor substrate binding region ofthe catalytic domain including the Cys-134-Cys-247 disulfidebond (50, 51). Although the latter bond might essentiallycontribute to a conformation required for interaction, it is notsufficient for binding as the snail ,84-GlcNAcT, which containsa homologous disulfide bond (44), is not responsive to a-LA asmentioned above. It has been reported that the ability of a-LAto bind to j34-GalT has to be distinguished from that topromote Glc binding, and a model has been proposed in whicha-LA interacts with 34-GalT directly by means of its so calledaromatic cluster I and indirectly via a Glc monosaccharidebridge (52). Whether a-LA likewise can interact with the snail,34-GalNAcT in this dual way is not known, but our data arenot incompatible with such a possibility. Clearly, the molecularcloning of the snail 634-GalNAcT is a crucial step to a betterunderstanding of the interaction of a-LA with both enzymes.The biological role of the interaction of L. stagnalis ,34-

GalNAcT with a-LA remains obscure. Neither the productGalNAc,B1--4Glc nor a-LA (38) have been described to occurin this species. Interestingly, the disaccharide unit has beenfound to constitute the core of sphingoglycolipids in adultworms (53) and eggs (54) of S. mansoni, but with the snailenzyme no activity was found with glucosylceramide as anacceptor (data not shown). It has been hypothesized thatlysozyme c, a lytic enzyme that degrades bacterial cell wallpeptidoglycans and that is homologous to a-LA (38, 39), in

protolacteal secretions helped protect the eggs or young frommicrobial infections (55). Presumably, the perivitelline fluidsecreted by the albumen gland of L. stagnalis contains thisprotein (56). However, neither lysozyme nor any proteinfraction isolated from the snail was capable of stimulating theaction of ,34-GalNAcT on Glc. Based on 5S ribosomal RNAsequence comparisons, the divergence of molluscs and verte-brates has been dated -500 million years ago (57). This datesvery long before the lysozyme gene duplication event that gaverise to Ca2+ binding forms of lysozyme from which the a-LAsdeveloped in mammals during evolution (38, 39). This raisesthe intriguing question of how the capability of the snail,B4-GalNAcT to interact with a-LA has evolved or has beenconserved during evolution in the absence of any apparentselective pressure. At present it is hard to answer this question,but it is of interest to note that the property of interacting witha-LA has also been conserved in f34-GalT from chicken,another nonlactating animal (46, 58), and in a plant f34-GalT(59).We would like to thank Mr. Carool Populier for providing us with

healthy specimens of L. stagnalis. The technical assistance of Ms.Carolien Koeleman and Ms. Angelique van Tetering is highly appre-ciated.

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