©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Lactalbumin Induces Bovine Milk 1,4-Galactosyltransferase to Utilize UDP-GalNAc (*)

(Received for publication, May 10, 1995; and in revised form, June 6, 1995)

Ki-Young Do Su-Il Do Richard D. Cummings (§)

From the University of Oklahoma Health Sciences Center, Department of Biochemistry and Molecular Biology, BSEB-325, Oklahoma City, Oklahoma 73190

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We now report that alpha-lactalbumin (alpha-LA) has a novel effect on bovine milk UDP-Gal:GlcNAc-beta1,4-galactosyltransferase (beta1,4-GT) and induces the enzyme to efficiently utilize UDP-GalNAc as a donor. In the presence of alpha-LA the enzyme transfers GalNAc to free GlcNAc to produce GalNAcbeta1-4GlcNAc at a rate 55% of that compared to the rate when UDP-Gal is the donor in the absence of alpha-LA. The stimulation by alpha-LA is dependent on the concentrations of alpha-LA, acceptor, and sugar nucleotide. Interestingly, beta1,4-GT is unable to transfer GalNAc to Glc with or without alpha-LA. alpha-LA also stimulates the transfer of GalNAc from UDP-GalNAc to various chitin oligomers, although the degree of stimulation decreases as the acceptor size increases. Thus, bovine milk beta1,4-GT has an inherent ability to utilize two different sugar nucleotides and the sugar nucleotide preference is regulatable by alpha-LA.


INTRODUCTION

Glycosyltransferases catalyze the transfer of sugar from a sugar nucleotide donor to an acceptor glycone or aglycone(1) . One of the best studied glycosyltransferases is lactose synthetase (EC 2.4.1.22), which is composed of a galactosyltransferase that transfers Gal from UDP-Gal to Glc in the presence of the regulatory protein alpha-lactalbumin (alpha-LA)(^1)(2, 3, 4) . The galactosyltransferase component of lactose synthetase is the enzyme, UDP-Gal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta1,4-GT) (EC 2.4.1.38). In non-lactating tissue, beta1,4-GT catalyzes the incorporation of galactose in beta1,4-linkage to GlcNAc residues at the nonreducing termini of certain glycoproteins and glycolipids(3, 5) . The enzyme can also efficiently transfer galactose to the monosaccharide GlcNAc to form the disaccharide N-acetyllactosamine (Galbeta1-4GlcNAc). It has been proposed that in addition to these biosynthetic functions, beta1,4-GT may also participate in cell adhesion (6) .

Glycosyltransferases have a preferred sugar nucleotide donor, and for beta1,4-GT this has been shown to be UDP-Gal. Several studies have indicated, however, that beta1,4-GT can slowly and inefficiently transfer glucose, arabinose, and N-acetylgalactosamine from their UDP derivatives to N-acetylglucosamine in vitro. For example, beta1,4-GT uses UDP-Glc and UDP-GalNAc at only 0.3 and 0.19%, respectively, of the rate at which it utilizes UDP-Gal (7, 8) .

In recent years we and others have described oligosaccharides in glycoproteins containing the terminal sequence GalNAcbeta1,4GlcNAcbeta1-R (9, 10, 11) . The biological functions of the sequence GalNAcbeta1-4GlcNAc-R in oligosaccharides are unclear, but it may be important for interactions between glycoproteins and specific receptors(12, 13) . The enzymes responsible for synthesizing the terminal beta1-4GalNAc structures have been found in several sources(14, 15, 16) , but there are many questions about the diversity of these enzymes and uncertainties as to whether the known beta1,4-GT might be responsible for some of the observed N-acetylgalactosaminyltransferase activities.

While assaying extracts of vertebrate cell lines for the presence of a glycosyltransferase that could transfer GalNAc from UDP-GalNAc to GlcNAc, we identified an activity apparently specific for UDP-GalNAc. In testing whether this activity might in fact be the known beta1,4-GT or related to it, we examined the effect of alpha-LA and conducted corresponding control experiments with bovine milk beta1,4-GT. During these studies we made the unexpected observation that beta1,4-GT was tremendously stimulated to use UDP-GalNAc as a donor and GlcNAc as the acceptor in the presence of alpha-LA. We now describe our studies of this unusual sugar nucleotide specificity of the beta1,4-GT in response to the regulatory protein alpha-LA.


EXPERIMENTAL PROCEDURES

Materials

UDP-Gal, UDP-GalNAc, UDP-Glc, NAD, MnCl(2), p-nitrophenyl sugar derivatives, UDP-Gal:GlcNAc beta1,4-GT from bovine milk (6.0 units/mg protein), nucleoside-5`-diphosphate kinase, uridine-5`-diphosphate glucose pyrophosphorylase, uridine-5`-diphosphate glucose dehydrogenase, ATP, alpha-LA, lysozyme, and jack bean beta-N-acetylhexosaminidase were obtained from Sigma. Dowex AG-1 X8 was purchased from Bio-Rad. UDP-[4,5-^3H]Gal (50 Ci/mmol) and UDP-[1-^3H]GalNAc (6.3 Ci/mmol) were purchased from DuPont NEN. Wisteria floribunda agglutinin-agarose (2 mg/ml) was purchased from Vector and concanavalin A-Sepharose was obtained from Pharmacia Biotech Inc.

Assay of beta1,4-GT

A radioactive assay for beta1,4-GT was conducted as described(17) . The reaction mixture normally contained the following materials in a total volume of 50 µl: bovine milk beta1,4-GT diluted to 2-10 milliunits/µl in phosphate-buffered saline containing 1.0 mg/ml bovine serum albumin, 20 mM MnCl(2), 5 mM ATP, 2.5-20 mMN-acetylglucosamine as the acceptor molecule, 10-20 µM UDP-[^3H]Gal (40,000-60,000 cpm/nmol) or similar amounts of UDP-[^3H]GalNAc, alpha-LA at different concentrations, and 10 mM sodium cacodylate buffer, pH 7.5. The reaction mixtures were incubated at 37 °C for 1-2 h and stopped by adding 1.0 ml of water and stored on ice. The product was isolated by passage of the reaction mixture over a 1.0-ml column of Dowex AG 1-X8 (chloride form) equilibrated with 5% borate in water (17) and washed with water to neutrality before each use. Each column was eluted 5 times with 2.0 ml of Milli-Q water and mixed with 2.0 ml of scintillation fluid and the amount of radioactivity was determined by liquid scintillation counting. Control incubations were done without an acceptor and the radioactivity eluting from the above column was subtracted as background. All assays were done in duplicate and basic statistical analysis was applied to the data. Acceptor p-nitrophenyl sugar derivatives were used at concentrations of 10 mM and products were isolated as described above on a column of Dowex AG 1-X8.

DEAE-Sephacel Chromatography and Spectrophotometric Assay for beta1,4-GT

The beta1,4-GT was suspended at 2 milliunits/µl in 100 mM cacodylate buffer, pH 7.5, containing 1.0 mg/ml bovine serum albumin. A DEAE-Sephacel column (1 10 cm) was equilibrated with 100 mM cacodylate buffer, pH 7.5. A total of 400 milliunits of enzyme was applied and the column was washed with 50 ml of equilibration buffer. The bound enzyme was eluted with a linear gradient of 0-300 mM NaCl gradient in equilibration buffer. Fractions (1 ml) were collected and assayed for Gal and GalNAc transferase activity by a spectrophotometric assay (18) . This assay measures the production of UDP through a coupled enzyme reaction leading to formation of NADH. The spectrophotometric assay consisted of the following components: 0.5 ml of 20 mM MnCl(2), 0.28 ml of 100 mM cacodylate buffer, pH 7.5, 0.01 ml of nucleoside-5`-diphosphate kinase (1000 units/ml), 0.01 ml of UDP-glucose pyrophosphorylase (25 units/ml), 0.01 ml of UDP-glucose dehydrogenase (2 units/ml), 0.067 ml of NAD (10 mg/ml), 0.05 ml of ATP (10 mg/ml), 0.02 ml of 5 mM UDP-Gal (or UDP-GalNAc), 0.02 ml of 1 MN-acetylglucosamine, and 200 µl of each fraction pool. After mixing, the tubes were incubated for 2 h at 37 °C and the absorbance at 340 nm was recorded in a Beckman spectrophotometer DU 640. For GalNAc transferase activity, UDP-GalNAc was added instead of UDP-Gal and alpha-LA (8 mg/ml) was included. Control assays were performed by conducting reactions in the absence of acceptor monosaccharide.

Lectin Affinity Column Chromatography

W. floribunda agglutinin-agarose (2 mg/ml) was placed in a column constructed from two 1-ml plastic disposable pipettes linked together (0.5 30 cm), and equilibrated in phosphate-buffered saline/NaN(3). The disaccharide product GalNAcbeta1-4GlcNAc was applied to the column and bound materials were eluted with 5 mM GalNAc in phosphate-buffered saline/NaN(3). Fractions (1 ml) were collected and radioactivity was determined by liquid scintillation counting.

Paper Chromatography and Exoglycosidase Treatment

Descending paper chromatography for separation of oligosaccharides was performed in solvent n-butanol:pyridine:water (6:4:3) for 72 h on borate-impregnated paper or ethyl acetate:pyridine:glacial acetic acid:water (5:5:1:3) for 24 h(19, 20) . The distribution of radioactivity on the paper chromatogram was determined by cutting the paper strips into 1-cm sections and measuring the radioactivity associated with each section in a liquid scintillation counter (Beckman). Treatment of oligosaccharides with jack bean beta-N-acetylhexosaminidase was performed with 20 milliunits of enzyme in 0.1 M sodium acetate, pH 5.6, for 20 h at 37 °C. The reaction mixtures were spotted on Whatman paper and analyzed again by descending paper chromatography.

Preparation of Chitin Oligomers and Amine-adsorption HPLC

Chitin oligomers were purified as described previously (21) and further purified by amine-adsorption HPLC on an AX5 column (4.0 mm 30 cm). The AX5 column was washed with water at a flow rate of 1.0 ml/min and equilibrated with 70% acetonitrile. The mixture of chitin oligomers (500 µg) was injected and 0.5 ml of fractions were collected using a linear gradient of 70 to 40% acetonitrile for 20 min. Each fraction was subsequently reanalyzed for purity by thin layer chromatography. By this procedure we purified N,N`-diacetylchitobiose, N,N`,N"-triacetylchitotriose, and N,N`,N",N-tetraacetylchitotetraose. The oligosaccharides were lyophilized and utilized as acceptors. The disaccharide product GalNAcbeta1-4GlcNAc was also analyzed using the same amine-adsorption HPLC system.


RESULTS

GalNAc Transfer by beta1,4-GT Is Stimulated by alpha-LA

Bovine milk beta1,4-GT transfers GalNAc to GlcNAc very poorly in vitro, but high amounts of the enzyme have been used to synthesize glycoconjugates terminating with GalNAcbeta1-4GlcNAc-R(8, 12) . We found, however, that the rate of reaction with UDP-GalNAc as a donor and free GlcNAc as an acceptor was greatly stimulated by alpha-LA (Fig. 1A). Low concentrations of alpha-LA had little effect, but higher concentrations above 5 mg/ml were stimulatory. Following this result, we used 8 mg/ml alpha-LA in routine assays. This stimulation of GalNAc transferase activity was dependent on the concentration of GlcNAc (Fig. 1B) and UDP-GalNAc (Fig. 1C). The K values of beta1,4-GT for GlcNAc and UDP-GalNAc were 21.6 mM and 52 µM, respectively, in the absence of alpha-LA. In the presence of alpha-LA the K values of beta1,4-GT for GlcNAc and UDP-GalNAc were 15.5 mM and 45 µM, respectively. alpha-LA increased the apparent V(max) at least 30-fold for UDP-GalNAc from approximately 0.8 to 23 pmol/h.


Figure 1: Dependence of N-acetylgalactosaminyltransferase activity of beta1,4-GT on alpha-LA. The reactivity of beta1,4-GT was measured as a function of alpha-LA (A), GlcNAc (B), and UDP-GalNAc (C). All enzyme assays were performed as described under ``Experimental Procedures.'' In B and C the alpha-LA concentration was 8 mg/ml and in A and C the GlcNAc concentration was 20 mM.



This striking change in sugar nucleotide specificity is best shown by data in Fig. 2, where the activity of beta1,4-GT for UDP-Gal and UDP-GalNAc are compared directly in the presence or absence of alpha-LA using GlcNAc as the monosaccharide acceptor. As expected, in the presence of alpha-LA, beta1,4-GT is inhibited in transferring Gal from UDP-Gal to GlcNAc. However, the activity of beta1,4-GT obtained with UDP-GalNAc as a donor was highly stimulated in the presence of alpha-LA and was approximately 55% of that obtained with UDP-Gal as a donor in the absence of alpha-LA.


Figure 2: Switch in sugar nucleotide specificity of beta1,4-GT by alpha-LA. Enzyme assays were performed as described under ``Experimental Procedures'' using GlcNAc (20 mM) as an acceptor, either UDP-[^3H]Gal or UDP-[^3H]GalNAc as sugar nucleotide donors (70 cpm/pmol), and beta1,4-GT (4 milliunits) in the absence and presence of alpha-LA (2 mg/ml).



Analysis of the Disaccharide Product

The product of the beta1,4-GT reaction using radioactive UDP-[^3H]GalNAc as a donor and GlcNAc as acceptor in the presence of alpha-LA was anticipated to be the disaccharide GalNAcbeta1-4GlcNAc. The product migrated as a single peak and moved slightly slower than N-acetyllactosamine during descending paper chromatography on borate-impregnated paper in the 6:4:3 solvent system but co-migrated with N-acetyllactosamine during descending paper chromatography on regular paper in the 5:5:1:3 solvent system (data not shown). These results indicate that the product is a disaccharide. To further confirm this, the disaccharide product was treated with jack bean beta-hexosaminidase and then re-analyzed by descending paper chromatography on borate paper. All radioactivity was recovered as the monosaccharide N-acetylgalactosamine following the exoglycosidase treatment. Upon analysis by amine-adsorption HPLC the disaccharide product eluted in the region where standard disaccharides elute (data not shown).

We also determined the affinity of the disaccharide toward the immobilized plant lectin W. floribunda agglutinin, a lectin previously shown to bind with high affinity to oligosaccharides containing terminal GalNAc in beta1,4-linkage to GlcNAc (22, 23) . All the disaccharide bound tightly to the lectin column and was eluted with 5 mM GalNAc (data not shown).

Taken together, these results demonstrate that the product is the disaccharide GalNAcbeta1-4GlcNAc. This conclusion is consistent with the results of others(8) , in which commercial beta1,4-GT was used to transfer GalNAc to GlcNAc-R, where R is a hydrophobic aglycone, albeit at a low rate given that alpha-LA was absent. In that study they also reported that the product formed with UDP-GalNAc as a donor and GlcNAc-R as an acceptor was GalNAcbeta1-4GlcNAc-R.

DEAE-Sephacel Chromatography of beta1,4-GT

To confirm that the single enzyme beta1,4-GT was able to efficiently transfer both GalNAc and Gal from UDP-GalNAc and UDP-Gal, respectively, we determined whether the Gal and GalNAc transferase activities co-eluted upon ion exchange column chromatography. beta1,4-GT (400 milliunits total; 6 units/mg) was suspended in 100 mM cacodylate buffer, pH 7.5, containing 1 mg/ml bovine serum albumin and was applied to a column of DEAE-Sephacel and eluted with a linear gradient of 0-300 mM NaCl. For convenience the activities of the enzyme were measured by a spectrophotometric assay(18) . In this assay the UDP produced by the enzyme from either UDP-Gal or UDP-GalNAc utilization is coupled to the production of NADH which is measured at an absorbance of 340 nm. Both the Gal and GalNAc transferase activities co-eluted with 70-100 mM NaCl during the gradient elution (data not shown). This elution behavior is consistent with previous reports(24) . UDP-Gal utilization was measured in the absence of alpha-LA, whereas UDP-GalNAc utilization was measured in the presence of alpha-LA. No significant activity was observed when UDP-GalNAc was used in the absence of alpha-LA. These results demonstrate that both the Gal and GalNAc transferase activities reside in beta1,4-GT.

Specific Effect of alpha-LA

alpha-LA and c-type lysozymes have 40% homology in amino acid sequence and are very similar in three-dimensional structure(25, 26) . Lysozyme degrades bacterial cell wall and does not bind to beta1,4-GT(27) . To test the specific effect of alpha-LA for enhancing GalNAc transfer, we added lysozyme to the enzyme reaction. GalNAc transferase activity toward GlcNAc was specifically stimulated by alpha-LA (11,534 cpm of product) but not by lysozyme (not significant cpm of product).

Lack of Transfer of GalNAc to Glc by beta1,4-GT

We further tested for acceptor specificity of beta1,4-GT in the presence of alpha-LA. As expected, alpha-LA caused the beta1,4-GT to efficiently utilize Glc as an acceptor with UDP-Gal as the donor (Table 1). However, there was no significant transfer of GalNAc to Glc in the presence of alpha-LA (Table 1).



Transfer of GalNAc to Oligosaccharides

We tested whether beta1,4-GT could transfer GalNAc from UDP-GalNAc to oligomers of GlcNAc and whether alpha-LA was also stimulatory for this reaction. alpha-LA stimulated the GalNAc transferase activity of the enzyme toward oligomers of GlcNAc up to N,N`,N",N-tetraacetylchitotetraose (GlcNAc)(4) (Table 2). The stimulatory effect declined, however, as the size of the oligosaccharide increased. Surprisingly, the enzyme transferred GalNAc to p-nitrophenyl-beta-D-GlcNAc (GlcNAcbeta-O-pNP) and to p-nitrophenyl-beta-D-chitobiose (GlcNAc-GlcNAc-beta-O-pNP) without added alpha-LA, although alpha-LA stimulated transfer somewhat with these acceptors (Table 2). As controls we tested the acceptors p-nitrophenyl-beta-D-xylose and p-nitrophenyl-beta-D-GalNAc and neither was efficiently utilized by beta1,4-GT with or without alpha-LA (data not shown).




DISCUSSION

Previous studies have shown that beta1,4-GT exhibits a marked preference for UDP-Gal as the sugar nucleotide donor and poorly utilizes alternative donors like UDP derivatives of glucose, N-acetylgalactosamine, deoxyglucose, and arabinose. The rates of reactions of beta1,4-GT with these other donors are only 0.2-4% as efficient as with UDP-Gal(8, 28) . Despite this poor reactivity with UDP-GalNAc, the beta1,4-GT has been used to enzymatically synthesize interesting glycoconjugates containing the terminal sequence GalNAcbeta1-4GlcNAc-R(8, 12) .

Until now, the major effect that alpha-LA has been considered to have on beta1,4-GT is to cause a switch in monosaccharide acceptor specificity from GlcNAc to Glc. The K of beta1,4-GT for Glc is reduced about a 1000-fold in the presence of alpha-LA from 2 M down to 2 mM(29, 30) . As already known, alpha-LA inhibits bovine beta1,4-GT from efficiently utilizing UDP-Gal to transfer Gal to GlcNAc. Our study shows, however, that alpha-LA induces the beta1,4-GT to switch to the alternative donor UDP-GalNAc when GlcNAc is the acceptor.

Many studies have been conducted on the kinetic mechanisms of beta1,4-GT and the effects of alpha-LA on enzyme activity and specificity. It has been proposed that beta1,4-GT operates by a partially ordered and sequential reaction mechanism(31, 32) , in which the enzyme binds first to Mn and UDP-Gal to form the EbulletMnbulletUDP-Gal complex, and this complex then interacts with either a monosaccharide acceptor GlcNAc or alpha-LA and N-acetyllactosamine synthesis is inhibited or stimulated depending on the relative concentrations of these latter two components. However, many aspects of the interaction of beta1,4-GT with alpha-LA and the specific mechanism of action of beta1,4-GT are poorly understood.

The location of regions in beta1,4-GT involved in binding to sugar nucleotide donor and alpha-LA have been studied by differential labeling and chemical modification(33, 34, 35) . Recent studies using site-directed mutagenesis(36) , revealed that alpha-LA has two aromatic clusters and that cluster I is involved in binding to beta1,4-GT. A model was proposed whereby alpha-LA binding to beta1,4-GT creates a ``monosaccharide bridge'' for the binding of glucose. Precisely how our finding on the switch to UDP-GalNAc as a donor in the presence of alpha-LA fits in with these observations is unknown. Interestingly, we found no evidence that beta1,4-GT can transfer GalNAc to Glc in either the presence or absence of alpha-LA.

The lack of transfer of GalNAc to Glc suggests that the conformation of the complex EbulletMnbulletUDP-GalNAc may be different from the conformation of the complex EbulletMnbulletUDP-Gal in that alpha-LA cannot expose the binding pocket for glucose in the former complex. Instead, the enzyme complex EbulletMnbulletUDP-GalNAc must still be capable of interacting with alpha-LA to promote transfer of GalNAc to GlcNAc. This implies that the GlcNAc-binding site is preserved in the complex EbulletMnbulletUDP-GalNAcbulletalpha-LA. Since beta1,4-GT has a single binding site for UDP, we presume that UDP-Gal and UDP-GalNAc occupy the same catalytic site. However, the effect of UDP-GalNAc on the enzyme complex with alpha-LA must be different from the effect of UDP-Gal, since alpha-LA blocks transfer of Gal from UDP-Gal to GlcNAc. It is also possible that alpha-LA could stimulate the GalNAc transfer activity of the enzyme by altering the rate of product release when GalNAc terminating products are synthesized. For example, it was previously observed that covalent cross-linking of alpha-LA with beta1,4-GT reduced the maximum velocity by a factor of 100 for lactose synthesis compared to the non-cross-linked enzyme for N-acetyllactosamine synthetase activity(37) . Many further experiments are needed to elucidate the precise mechanism by which alpha-LA causes the sugar nucleotide switch of beta1,4-GT.

Some glycosyltransferases exhibit a lower K for acceptors containing a hydrophobic aglycone than for the free oligosaccharide acceptor(38, 39) . This may relate to our observation that p-nitrophenyl-beta-D-GlcNAc was more efficient than free GlcNAc as an acceptor for the GalNAc transferase activity of beta1,4-GT even without added alpha-LA (Table 2). As mentioned above, it has been suggested that aromatic amino acids in alpha-LA are important for both alpha-LA interaction with beta1,4-GT and induction of the substrate glucose binding pocket, and these two sites are in close proximity(36) . Our results suggest the possibility that the aromatic aglycone moiety of the acceptor p-nitrophenyl-beta-D-GlcNAc might substitute for the aromatic cluster of alpha-LA and thereby stimulate beta1,4-GT to transfer GalNAc to GlcNAc in the absence of alpha-LA. This phenomenon needs further study, since the nature of the aglycone may be important and aromatic aglycone derivatives may be more active in this regard than non-aromatic aglycones. For example, it has been observed (8) that beta1,4-GT could transfer GalNAc from UDP-GalNAc to the 8-methoxycarbonyloctyl hydrophobic aglycone derivative of GlcNAc, but the rate was 0.19% of that obtained when UDP-Gal was the donor.

In preliminary experiments we found that beta1,4-GT could transfer GalNAc from UDP-GalNAc to biantennary acceptor glycopeptides and the reaction was stimulated by alpha-LA, but the degree of stimulation was not as great as observed with the chitin-like acceptors. This may result from the large size of the acceptors that interfere with alpha-LA binding to the beta1,4-GT, as previously observed for other acceptors when UDP-Gal is the donor(32, 40, 41) . We are currently examining in more detail the interesting effect of alpha-LA on the ability of beta1,4-GT to transfer GalNAc to glycoprotein acceptors. In addition, we are studying whether this effect of alpha-LA extends to glycosphingolipid acceptors.

Several glycoproteins from bovine milk, including bovine alpha-LA, bovine lactotransferrin, and bovine milk epithelial glycoprotein IV, contain oligosaccharides with the terminal sequence GalNAcbeta1-4GlcNAc-R (42, 43, 44, 45) . Terminal beta1,4-linked GalNAc is a common residue in Asn-linked oligosaccharides in many membrane glycoproteins from bovine milk fat globule (45) and these glycoproteins may acquire more GalNAc during lactation(46) . Our results suggest the possibility that under certain conditions within the lactating mammary gland, where the concentration of alpha-LA is high, the beta1,4-GT might be able to generate glycoproteins containing terminal GalNAc structures.

Terminal GalNAcbeta1-4GlcNAc-R has been found in many other non-mammary gland-derived glycoproteins(47, 48, 49, 50, 51, 52, 53, 54) and in glycoproteins derived from the parasites Schistosoma mansoni and Dirofilaria immitis(11, 55) . A beta1,4-N-acetylgalactosaminyltransferase distinct from beta1,4-GT has been shown to be important in the synthesis of terminal beta1,4-GalNAc structures in pituitary glycoprotein hormones(14) , but other N-acetylgalactosaminyltransferase activities have also been reported(15, 16) . The degree to which the effects we have observed on beta1,4-GT are related to the synthesis of terminal beta1,4-GalNAc structures is unknown. alpha-LA is highly expressed in the lactating mammary gland where its expression is associated with lactose production, but the UDP-GalNAc utilization induced by alpha-LA is unrelated to lactose production, since Glc is not an acceptor when UDP-GalNAc is the donor. Whether there are other modifier proteins expressed by non-mammary gland-derived cells, capable of mimicking alpha-LA and altering the sugar nucleotide specificity of beta1,4-GT, remains to be determined. In addition, we do not know whether the effect we have observed is specific only for beta1,4-GT or whether some other glycosyltransferases may be induced to use alternative sugar nucleotide donors in the presence of alpha-LA or some modifier proteins or factors.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA-37626 (to R. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The University of Oklahoma Health Sciences Center, Dept. of Biochemistry and Molecular Biology, P. O. Box 26901, BSEB-325, Oklahoma City, OK 73190. Tel.: 405-271-2546; Fax: 405-271-3910.

^1
The abbreviations used are: alpha-LA, alpha-lactalbumin; beta1,4-GT, UDP-Gal:beta-D-GlcNAc beta-1,4-galactosyltransferase; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Gal, galactose; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. A. Kwame Nyame for critically reading the manuscript.


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