Bovine mammary gland UDP-GalNAc:GlcNAc[beta]-R [beta]1->4-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with [alpha]-lactalbumin

Ingrid M. Van den Nieuwenhof, Wietske E.C.M. Schiphorst, Irma Van Die and Dirk H. Van den Eijnden1

Department of Medical Chemistry, Faculty of Medicine, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

Received on March 4, 1998; revised on June 15, 1998; accepted on July 5, 1998

We have identified a novel N-acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz 1H-NMR spectroscopy revealed that the enzyme catalyses the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, [beta]-linked N-acetylglucosamine (GlcNAc) residue and establishes a [beta]1->4-linkage forming a GalNAc[beta]1->4GlcNAc (N,N[prime]-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAc[beta]-R [beta]1->4-N-acetylgalactosaminyltransferase ([beta]4-GalNAcT). This enzyme resembles invertebrate [beta]4-GalNAcT as well as mammalian [beta]4-galactosyltransferase ([beta]4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific [beta]4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif. Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a [beta]4-GalT as could be demonstrated by comparing the [beta]4-GalNAcT and [beta]4-GalT activities of the mammary gland, bovine colostrum, and purified [beta]4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-[beta]4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian [beta]4-GalT forms with [alpha]-lactalbumin ([alpha]-LA) the lactose synthase complex, the mammary gland [beta]4-GalNAcT was similarly induced by [alpha]-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAc[beta]1->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland [beta]4-GalNAcT functions in the synthesis of lacdiNAc-based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.

Key words: N-acetylgalactosaminyltransferase/N,N[prime]-diacetyllactosediamine/milk glycoproteins/[beta]4-galactosyltransferase family/complex-type glycans

Introduction

Glycoprotein glycans typically contain Gal[beta]1->4GlcNAc (N-acetyllactosamine, lacNAc) units, which serve as backbone structural elements. These backbone structures, which are built on an N- or O-linked core structure, are the carriers of terminal carbohydrate epitopes, such as sialyl-Lewisx, that often confer a specific biological property (e.g., blood group activity) on the glycoprotein. In a considerable number of instances, however, a variant of this backbone structure has been described, in which the Gal residue is replaced by a GalNAc, resulting in a GalNAc[beta]1->4GlcNAc (N,N[prime]-diacetyllactosdiamine, lacdiNAc) unit (see references cited in Van den Eijnden et al., 1997). In vertebrates the group of glycoproteins that carry lacdiNAc-based chains is quite diverse and comprises membrane as well as secreted glycoproteins, enzymes, hormones, transport proteins, and protective glycoproteins (Green and Baenziger, 1988; Coddeville et al., 1992; Sato et al., 1993; Bergwerff et al., 1995; Hooper et al., 1995; Taguchi et al., 1995). Interestingly, bovine milk glycoproteins are particularly rich in lacdiNAc-based complex-type glycans (Tilley et al., 1991; Coddeville et al., 1992; Nakata et al., 1993; Sato et al., 1993, 1995; Aoki et al., 1995; Girardet et al., 1995), indicating that bovine mammary gland possesses a high potential to produce such glycans.

LacdiNAc-based glycans of mammalian glycoproteins have been proposed to be specific ligands for certain cellular lectins thus serving a biological function. For instance the 4[prime]-O-sulfated-lacdiNAc epitope occurring on the glycans of lutropin is recognized by a receptor on hepatic endothelial and Kupffer cells resulting in a functional rapid half-life of this glycohormone in the circulation (Fiete et al., 1991). The [alpha]3-fucosylated form of lacdiNAc present on the glycans of recombinant protein C has been suggested to confer anti-inflammatory properties on this anticoagulant factor by being a ligand for E-selectin on vascular endothelium (Grinnell et al., 1994). Furthermore, the immunosuppressive effect of glycodelin A has been proposed to be mediated by the recognition of [alpha]6-sialylated-lacdiNAc termini by CD22 on B cells, whereas the same epitope and/or [alpha]3-fucosylated-lacdiNAc on this glycoprotein is believed to be implicated in the contraceptive properties of glycodelin A, through its interaction with a lectin at the surface of spermatocytes or oocytes (Dell et al., 1995). So far, however, no glycan-dependent function has been reported for the lacdiNAc-type glycoproteins present in bovine milk.

A UDP-GalNAc:GlcNAc[beta]-R [beta]4-N-acetylgalactosaminyltransferase ([beta]4-GalNAcT) involved in the synthesis of the lacdiNAc-based, N-linked glycans on hormones like lutropin and thyrotropin has been described in bovine pituitary gland (Smith and Baenziger, 1988). This enzyme has been suggested to specifically recognize a peptide motif that is present in glycoprotein hormones and to act with a 100- to 200-fold greater catalytic efficiency on glycoprotein substrates carrying this motif (Mengeling et al., 1995; Manzella et al., 1996). By contrast, in invertebrates such as schistosomes, snails and insects, the synthesis of lacdiNAc units is controlled by a peptide motif independent [beta]4-GalNAcT (Neeleman et al., 1994; Srivatsan et al., 1994; Mulder et al., 1995; Van Die et al., 1996). This [beta]4-GalNAcT differs from the pituitary gland enzyme in that it acts on any substrate exposing a nonreducing, terminally [beta]-linked GlcNAc residue regardless of the underlying structure.

In this study we have identified and characterized a [beta]4-GalNAcT in bovine mammary gland that is concluded to be involved in the synthesis of the lacdiNAc units on bovine milk glycoprotein glycans. This enzyme resembles the invertebrate [beta]4-GalNAcT in acceptor specificity and differs from the pituitary enzyme in its indifference to the glycoprotein-hormone-specific peptide motif. In addition, it appears to be responsive to [alpha]-lactalbumin ([alpha]-LA) and can act on Glc in the presence of this modifier.

Results

GalNAcT and GalT activity in bovine mammary gland


Figure 1. Inhibition of GalT activity (solid circles) and GalNAcT activity (solid squares) by a polyclonal antiserum directed against highly purified bovine thymus [beta]4-GalT (Blanken and Van den Eijnden, 1985). Standard incubation conditions were employed using 1 mM GlcNAc-S-pNP as an acceptor to assay the activity of the enzymes, except for the addition of varying amounts of antiserum.


Figure 2. Competition of GalT and GalNAcT of bovine mammary gland for the common acceptor substrate GlcNAc-S-pNP. The acceptor concentration was kept at 1 mM whereas the concentrations of UDP-Gal and UDP-GalNAc varied as indicated. The experimental points (solid circles) are the sum of the activities of GalT and GalNAcT as determined in two separate assays using UDP-[3H]Gal plus UDP-GalNAc, and UDP-Gal plus UDP-[3H]GalNAc, respectively, employing otherwise standard incubation conditions. The theoretical line for the case of a single enzyme being capable of transferring both Gal and GalNAc from the respective UDP-sugars was calculated from equation: vt = (VA·[A]·KmA-1 + VB·[B]·KmB-1)(1 + [A]·KmA-1 + [B]·KmB-1)-1 where vt is the total reaction velocity, and VA and VB the maximal reaction velocities, [A] and [B] the actual concentrations, and KmA and KmB the Km values for UDP-Gal and UDP-GalNAc, respectively. The theoretical line for the case of two enzymes, one specific for UDP-Gal, the other for UDP-GalNAc, was calculated from equation: vt = VA(1 + KmA·[A]-1)-1 + VB(1 + KmB·[B]-1)-1. The actual kinetic parameters for UDP-Gal and UDP-GalNAc where estimated to be V = 5.5 and 0.6 nmol·min-1·mg-1 protein, and Km = 0.27 and 0.24 mM, respectively.

In bovine mammary gland a GalNAcT activity was found amounting about 9% of the [beta]4-GalT activity using GlcNAc-S-pNP as an acceptor substrate (Table I). By contrast, in bovine colostrum, the fluid secreted by the gland, the GalNAcT activity amounted less than 1% of the GalT activity (Table I). Similarly, [beta]4-GalT highly purified from bovine colostrum showed a very low GalNAcT activity (Table I). Preincubation of bovine mammary gland membranes with 5 µl of a polyclonal antiserum directed against bovine [beta]4-GalT resulted in a 70% decrease in GalT activity with hardly any effect on the GalNAcT activity (Figure 1). The GalNAcT activity was only inhibited when substantially higher amounts of antiserum were used. Competition studies further demonstrated that the transfer of Gal and GalNAc from the respective UDP-sugar donors to GlcNAc-S-pNP catalyzed by bovine mammary gland membranes was the result of the action of two different enzymes. Incubations were conducted in which UDP-Gal and UDP-GalNAc were present in the same mixture. The experimental points were close to the values calculated for the case of two different enzymes each utilizing a different UDP-sugar, and differed from the values calculated for the case of a single enzyme capable of using UDP-Gal as well as UDP-GalNAc (Figure 2). A very similar result was obtained in a competition experiment with UDP-Gal and UDP-GalNAc as donors and Glc as acceptor in the presence of 12 mg·ml-1 [alpha]-LA (result not shown).

Table I. . Comparison of the [beta]4-GalT and [beta]4-GalNAcT activities of bovine mammary gland membranes, bovine colostrum and [beta]4-GalT purified from bovine colostrum
Enzyme source [beta]4-GalT activity
(nmol·min-1·mg-1)
[beta]4-GalNAcT activity
(nmol·min-1·mg-1)
Activity ratio
(GalNAcT/GalT, %)
Mammary gland 3.95 0.36 9.1
Colostrum 0.84 0.005 0.6
Purified [beta]4-GalT 6.1 × 103 24.5 0.4
Standard assays were performed using GlcNAc-S-pNP as an acceptor at a concentration of 1 mM as described under Materials and methods using assay method 2.

Acceptor substrate specificity of bovine mammary gland GalNAcT

The acceptor substrate specificity of the GalNAcT in bovine mammary gland membranes was investigated with linear oligosaccharides (with or without hydrophobic aglycon), branched oligosaccharides, and glycoprotein acceptor substrates (Table II). The results show that substrates carrying a terminal GlcNAc residue, including free GlcNAc (compound 1), can serve as an acceptor. Comparison of the activities of acceptor 8 with compounds 9 and 10 (Table II) demonstrates that a [beta]-configuration of this GlcNAc is essential for activity. Substrates with a terminal GalNAc residue, in either an [alpha]- or [beta]-configuration (compounds 14 and 15), could not serve as an acceptor. Highest activities were obtained with two oligosaccharide acceptor substrates having a hydrophobic aglycon (S-pNP and O-(CH2)8COO-CH3, compounds 10 and 12, respectively). Interestingly, when GlcNAc was linked O-glycosidically to pNP (acceptor 9) rather than by an S-glycosidic bond (as in compound 10), or linked to hydrophobic methylumbelliferon (11), a much lower activity was obtained. Activities obtained with chitobiose and chitotriose (compounds 6 and 7) were comparable with that obtained with GlcNAc. The penultimate sugar of the different disaccharide acceptors had no great influence on the activity. Depending on whether GlcNAc was linked to carbon 2, 3, 4, or 6 of Gal (2 and 3), Man (4 and 5) or GlcNAc (6), the activities maximally varied 3-fold. Similarly, the activities obtained with the branched oligosaccharide substrates (compounds 16, 17, 19-22) were similar to those obtained with the linear oligosaccharides. When contained in a protein-linked glycan (23,24), however, lower activities were observed, while the glycopeptide acceptor (as/ag-GP-F2, 18) yielded an activity comparable to that obtained with the free oligosaccharides.

To investigate whether the GalNAcT of bovine mammary gland resembles the [beta]4-GalNAcT of bovine pituitary gland (Smith and Baenziger, 1992) in being capable of recognizing a specific peptide motif in glycoprotein hormone acceptors, the activity with glycopeptide as/ag-GP-F4 and as/ag-hCG[alpha] (a glycoprotein acceptor containing the peptide motif) at two acceptor concentrations was compared (Table III). With 0.5 mM as/ag-GP-F4 a 7- to 8-fold higher activity was obtained than with 0.5 mM hCG[alpha]. At a 20-fold reduced concentration, however, no activity above the detection limit was found with either acceptor. Thus, unlike the pituitary [beta]4-GalNAcT, the mammary gland enzyme does not show an enhanced activity with acceptors containing the peptide motif and apparently does not posses a high affinity for such acceptors.


Figure 3. Effect of the concentration of [alpha]-LA on the incorporation of GalNAc from UDP-GalNAc into GlcNAc (5 mM) (solid circles) and Glc (50 mM) (solid squares) ,respectively, catalyzed by bovine mammary gland membranes. Otherwise standard incubation conditions as described under Materials and methods were employed.

Product characterization by 400 MHz 1H-NMR spectroscopy

Table II. Acceptor specificity of [beta]4-GalNAcT from bovine mammary gland
Relative activity was assayed at an acceptor concentration of 1 mM as described under Materials and methods; 100% activity corresponds to an activity of 0.024 nmol·min-1·mg-1 protein.

The partial 400 MHz 1H-NMR spectrum of the product formed by the action of the bovine mammary gland GalNAcT with GlcNAc as an acceptor is shown in Figure 3a. The numeric spectroscopic data are presented in Table IV. The spectrum shows H-1 and CH3-NAc signals typical for a reducing GlcNAc residue and a nonreducing GalNAc in [beta]-configuration. The spectral data of the product are virtually identical to those of GalNAc[beta]1->4GlcNAc published before (Nemansky and Van den Eijnden, 1992). It thus can be concluded that the structure of the enzymatic product is GalNAc[beta]1->4GlcNAc. Hence, it appears that the mammary gland enzyme is an UDP-GalNAc:GlcNAc[beta]-R [beta]1->4-N-acetylgalactosaminyltransferase.

Effect of [alpha]-LA on [beta]4-GalNAcT activity and specificity

In view of the analogy in the reactions catalyzed by [beta]4-GalT and the mammary gland [beta]4-GalNAcT and the known effect of the modifier [alpha]-LA on the activity and specificity of the former glycosyltransferase, we investigated the effect of [alpha]-LA on the [beta]4-GalNAcT. In the absence of [alpha]-LA the activity of the [beta]4-GalNAcT with GlcNAc (5 mM) amounted 5.2 mU/ml, whereas there was no detectable activity obtained with Glc (100 mM). Addition of [alpha]-LA induced the bovine mammary gland [beta]4-GalNAcT to act on Glc in addition to GlcNAc (Figure 4). The enzyme activity with GlcNAc as an acceptor increased almost 3-fold with increasing concentrations of [alpha]-LA (added from 2 to 10 mg·ml-1). By contrast, at higher concentrations of GlcNAc (>25 mM), no effect of [alpha]-LA was observed, but no inhibition of [beta]4-GalNAcT by [alpha]-LA was found at any concentration of GlcNAc tested (data not shown). Activity of the GalNAcT with Glc as an acceptor substrate was essentially dependent on the presence of [alpha]-LA. Addition of [alpha]-LA in concentrations above 2 mg·ml-1 to the incubation mixtures resulted in a GalNAcT activity of about 2 mU/ml (Figure 4).

To analyze the effect of [alpha]-LA on bovine mammary gland [beta]4-GalNAcT in more detail the Km and V values for GlcNAc and Glc in the absence and presence of 12 mg·ml-1 [alpha]-LA were determined (Table V). Addition of [alpha]-LA resulted in a 4-fold decreased Km for GlcNAc, and a 11-fold decrease in the Km for Glc. Since [alpha]-LA had little effect on the V values observed with either GlcNAc or Glc, the kinetic efficiencies for GlcNAc and Glc were increased 3.5- and 10-fold, respectively, upon addition of the modifier protein.

The partial 400 MHz 1H-NMR spectrum and the chemical shift values of the product formed with Glc by the action of the bovine mammary gland GalNAcT in the presence of [alpha]-LA are shown in Figure 3b and Table IV, respectively. The spectrum shows H-1 resonances and a single CH3-NAc signal typical for a nonreducing GalNAc in [beta]-configuration to a residue in anomeric equilibrium. Also, a double doublet characteristic of the H-2 of a [beta]-Glc residue was observed. Because the spectrum and chemical shift values are essentially identical to those of authentic GalNAc[beta]1->4Glc (Neeleman and Van den Eijnden, 1996), it can be concluded that the structure of the product formed was GalNAc[beta]1->4Glc.


Figure 4. Partial 400 MHz 1H-NMR spectra showing the structural reporter-group regions of the disaccharide products formed by incubating bovine mammary gland membranes with (A). UDP-GalNAc and GlcNAc and (B). UDP-GalNAc and Glc in the presence of [alpha]-LA.

Table III. . Comparison of the activity of [beta]4-GalNAcT from bovine mammary gland with glycopeptide asialo/agalacto-GP-F4 and asialo/agalacto-hCG[alpha] at two concentrations
Acceptor Concentration (mM) [beta]4-GalNAcT activity (pmol·min-1·mg-1 protein)
as/ag-GP-F4 0.025 <0.06
0.5 3.0
as/ag-[alpha]hCG 0.025 <0.06
0.5 0.4

Discussion

In this study, we have identified and characterized a UDP-GalNAc:GlcNAc[beta]-R [beta]1->4-N-acetylgalactosaminyltransferase ([beta]4-GalNAcT) in lactating bovine mammary gland that acts on virtually any acceptor with a terminal nonreducing GlcNAc residue regardless of the underlying structure. By a number of criteria, the [beta]4-GalNAcT can be clearly distinguished from the [beta]4-GalT, which is also present in the gland. Competition experiments showed that the GalNAcT activity is not due to a flexibility in donor substrate usage of the [beta]4-GalT (Palcic and Hindsgaul, 1991) but is due to a separate enzyme species. Moreover, at UDP-sugar concentrations [le]0.5 mM (as used in this study), donor promiscuity displayed by [beta]4-GalT has appeared to amount less than 1% (Palcic and Hindsgaul, 1991; Do et al., 1995; Neeleman and Van den Eijnden, 1996; this study) and therefore cannot account for the GalNAcT activity in mammary gland membranes. Furthermore, the two activities in mammary gland showed a differential responsiveness toward an inhibiting antibody raised to bovine [beta]4-GalT. Also, it could be excluded that the GalNAcT activity is due to the mammary gland [beta]4-GalT being induced to utilize UDP-GalNAc by [alpha]-LA (Do and Cummings, 1995) since the membrane preparation used was essentially free of [alpha]-LA. Thus the [beta]4-GalNAcT represents a separate, novel enzyme with an acceptor and linkage specificity that closely resembles that documented for [beta]4-GalT (Schachter and Roseman, 1980; Blanken et al., 1982, 1984; Narasimhan et al., 1985).

Several other [beta]4-GalNAcTs have been described to date, for example [beta]4-GalNAcTs involved in the synthesis of ganglio-series glycosphingolipids (Pohlentz et al., 1988) and the Sda blood-group determinant (Soh et al., 1989), and the [beta]4-GalNAcT that catalyses the key step in the synthesis of chondroitin sulfate (Rohrmann et al., 1985). The [beta]4-GalNAcT of bovine mammary gland differs from these enzymes because none of them act on acceptors containing a terminal [beta]-linked GlcNAc residue. Clearly, the mammary gland enzyme also differs from the pituitary [beta]4-GalNAcT (Smith and Baenziger, 1988), although both enzymes catalyze the synthesis of lacdiNAc-units on protein-linked, complex-type glycans. Unlike the pituitary enzyme the mammary gland [beta]4-GalNAcT is not glycoprotein-hormone-specific and does not act on a substrate carrying the hormone-specific peptide motif with an elevated efficiency. The specificity of the [beta]4-GalNAcT of bovine mammary gland rather resembles that of the [beta]4-GalNAcTs described in the albumen gland of the snail Lymnaea stagnalis (Mulder et al., 1995), cercariae of the schistosome Trichobilharzia ocellata (Neeleman et al., 1994), adult worms of Schistosoma mansoni (Srivatsan et al., 1994), and lepidopteran insect cell lines (Van Die et al., 1996), and might also be similar to that of a GalNAcT found in human embryonic kidney and in several malignant cell lines (Dharmesh et al., 1993). Also for these enzymes a terminal [beta]-linked GlcNAc appears to be a sufficient acceptor requirement.

In bovine milk a large number of glycoproteins have been described carrying lacdiNAc-based, complex-type N-linked glycans (reviewed in Van den Eijnden et al., 1997; see Introduction). The [beta]4-GalNAcT of bovine mammary gland clearly seems to function in the synthesis of these glycans. Together with the [alpha]6-sialyltransferase, that is highly expressed in the gland, this tissue contains the enzymatic potential (Nemansky and Van den Eijnden, 1992) to form the NeuAc[alpha]2->6GalNAc[beta]1->4GlcNAc termini that frequently occur on these glycoproteins (Coddeville et al., 1992; Nakata et al., 1993; Girardet et al., 1995). It is not known whether the human mammary gland expresses the [beta]4-GalNAcT and produces glycoproteins with lacdiNAc-based glycans. The notion that this enzyme is present in bovine mammary gland, however, is of special interest not only in view of attempts to humanize bovine milk to improve infant formulas (Maga and Murray, 1995; Rosen et al., 1996), but also with respect to the production of properly glycosylated glycoprotein therapeutics in the milk of transgenic mammals (Houdebine, 1995; Colman, 1996; Young et al., 1997).

Table IV. . NMR-data of the products (GalNAc[beta]1->4GlcNAc and GalNAc[beta]1->4Glc) formed by the action of bovine mammary gland [beta]4-GalNAcT on GlcNAc in the absence of [alpha]-LA and Glc in the presence of this modifier protein
Reporter group Residue Anomer of compound Chemical shift (coupling constant)
Product with GlcNAc Reference GalNAc[beta]1->4GlcNAca Product with Glc Reference GalNAc[beta]1->4Glcb
(ppm) (Hz) (ppm) (Hz) (ppm) (Hz) (ppm) (Hz)
H-1 Glc [alpha] - - - - 5.206 3.8 5.205 3.8
[beta] - - - - 4.651 8.1 4.650 8.0
H-2 Glc [beta] - - - - 3.263 9.0 3.263 9.3
H-1 GlcNAc [alpha] 5.193 2.9 5.191 2.8 - - - -
[beta] 4.702 8.2 4.701 8.3 - - - -
H-1 GalNAc [alpha] 4.531 8.4 4.530 8.4 4.512 8.5 4.511 8.5
[beta] 4.521 8.4 4.521 8.5 4.504 8.4 4.503 8.4
NAc-CH3 GlcNAc [alpha]/[beta] 2.037 - 2.037 - - - - -
NAc-CH3 GalNAc [alpha]/[beta] 2.067 - 2.067 - 2.063 - 2.063 -
Coupling constants represent J1,2 values except for H-2 of Glc (J2,3).
aData taken from Neeleman et al., 1994.
bData taken from Neeleman and Van den Eijnden, 1996.

Table V. . Effect of [alpha]-LA on the kinetic parameters of bovine mammary gland [beta]4-GalNAcT
Acceptor substrate Km (mM) V (nmol·h-1·mg-1protein) Kinetic efficiency (V·Km-1)
- + - + - +
GlcNAc 9.7 2.3 12.3 10.6 1.26 4.60
Glc 450 41 3.6 3.4 0.008 0.082
Assays were performed in the absence (-) or presence (+) of 12 mg·ml-1 [alpha]-LA using UDP-GalNAc as donor substrate.

Interestingly, bovine mammary gland [beta]4-GalNAcT appears to be responsive to [alpha]-LA. This modifier protein, that is produced in the lactating mammary gland, induced the enzyme to act on Glc in vitro with a 10-fold increased efficiency to form GalNAc[beta]1->4Glc. This result further distinguishes the [beta]4-GalNAcT from the [beta]4-GalT, since the latter enzyme cannot be induced by [alpha]-LA to transfer GalNAc to Glc (Do and Cummings, 1995). The mammary gland [beta]4-GalNAcT shares its responsiveness to [alpha]-LA with mammalian (Schanbacher and Ebner, 1970; Hill and Brew, 1975), marsupial (Messer and Nicholas, 1991), avian (Hathaway et al., 1991; Shaper et al., 1997), and plant (Powell and Brew, 1974) [beta]4-GalTs as well as with a [beta]4-GalNAcT found in the albumen gland of the snail L.stagnalis (Neeleman and Van den Eijnden, 1996). The mammary gland [beta]4-GalNAcT thus forms the second example of a mammalian enzyme the specificity of which can be modified by another protein. A noticeable difference in the effect of [alpha]-LA on the mammary gland and snail [beta]4-GalNAcTs as compared to the [beta]4-GalTs, however, is that [alpha]-LA induces the [beta]4-GalNAcTs to act also on GlcNAc with a higher efficiency, while the activities of the [beta]4-GalTs with this acceptor substrate (at concentrations >2-3 mM) were reported to be strongly reduced in the presence of [alpha]-LA.

For long it was assumed that all [beta]4-GalTs, with a [beta]4-GalT in porcine trachea as a notable exception (Sheares and Carlson, 1984), were responsive to [alpha]-LA. More recently, however, a [beta]4-GalT in S.mansoni was found to be nonresponsive to [alpha]-LA too (Rivera Marrero and Cummings, 1990). In addition the recent cloning of novel human [beta]4-GalTs (Almeida et al., 1997; Sato et al., 1998), which show varying sequence similarity to the human [beta]4-GalT cloned first (Masri et al., 1988), revealed the existence of more [beta]4-GalTs that are insensitive to [alpha]-LA. While human [beta]4-GalT 1 and [beta]4-GalT 2 interact with [alpha]-LA, human [beta]4-GalT 3 and 4 (see Almeida et al., 1997, and Sato et al., 1998, for the numbering system) are not sensitive to this modifier protein. Also the specificity of the related snail [beta]4-GlcNAcT (Bakker et al., 1994) was not altered by [alpha]-LA (Bakker et al., 1997). Among the [beta]4-GalNAcTs only the enzyme in T.ocellata has been reported to be indifferent to [alpha]-LA (Neeleman et al., 1994). It is not known how [beta]4-GalT interacts with [alpha]-LA, but comparison of the amino acid sequences of members of the [beta]4-galactosyltransferase gene family (Van Die et al., 1997) that are interacting with this modifier protein with those of nonresponsive members might aid in the understanding of this interaction.

A number of groups have presented structural data indicating that [beta]4-GalT and [beta]4-GalNAcT activities must coexist in several cells and tissues of higher animals (Baenziger and Green, 1988: Weisshaar et al., 1990, 1991; Hård et al., 1992; Hiyama et al., 1992; Siciliano et al., 1993; Smith et al., 1993; Tomiya et al., 1993; Yan et al., 1993). For instance ovine lutropin (Baenziger and Green, 1988; Weisshaar et al., 1990) and human Tamm-Horsfall glycoprotein (Hård et al., 1992) carry N-linked glycans that contain Gal[beta]1->4GlcNAc (lacNAc) as well as GalNAc[beta]1->4GlcNAc (lacdiNAc) structural elements on different branches. In addition, recombinant human protein C, expressed in human kidney cells, was also shown to contain lacdiNAc- as well as lacNAc-based chains in its Asn-linked oligosaccharides. When concurrently expressed and targeted to the same locale in the Golgi, the [beta]4-GalNAcT may compete with the [beta]4-GalT for common GlcNAc acceptor sites. This would imply that the relative expression levels along with the (branch)specificities of these enzymes determine to which extent lacNAc- and lacdiNAc-type chains are formed. The characterization of the [beta]4-GalNAcT of bovine mammary gland in this study contributes to our understanding of the controlling role of the enzyme in the lacdiNAc-pathway (Bakker et al., 1997; Van den Eijnden et al., 1997) of complex-type N-linked glycan synthesis. The molecular cloning of this enzyme will reveal whether or not it is genetically related to members of the [beta]4-galactosyltransferase family.

Materials and methods

Materials

The following persons kindly supplied us with oligosaccharides (the numbers refer to the structures given in Table II). Compounds 2 and 3 from Dr. L.Anderson (University of Wisconsin, Madison, Wisconsin); 4, 5, 16-17, and the [beta]4-galactosylated form of 20 from Dr. J.Lönngren (University of Stockholm, Stockholm, Sweden); 12 from Dr. O.Hindsgaul (University of Alberta, Edmonton, Alberta, Canada); the [beta]4-galactosylated form of 19 from Dr. G.Strecker (Université des Sciences et Techniques de Lille Flandres-Artois, Villeneuve d'Ascq, France); 21 fromDr. A.Veyrières (Université Paris Sud, Orsay, France); 22 from Dr. M.Haraldsson (University of Stockholm, Stockholm, Sweden). Compounds 1, 6-11, and 14-15 were obtained from Sigma. Compound 13 was obtained from Toronto Research Chemicals (Toronto, Ontario). The [beta]4-galactosylated oligosaccharides were degalactosylated as described below.

[alpha]1-Acid glycoprotein ([alpha]1-AGP) was isolated from human plasma Cohn fraction V supernatant according to Hao and Wickerhauser (1973). Fetuin from calf serum was obtained from Gibco (Paisley, Scotland). The [alpha]-subunit of human chorionic gonadotrophin (hCG[alpha]) was kindly donated by Dr. R.A.Wijnands (Diosynth B.V., Oss, The Netherlands). The glycoproteins were desialylated by mild acid treatment (0.1 M trifluoroacetic acid, 1 h, 80°C) to give the asialo (as) forms. Diantennary glycopeptides GP-F2 and GP-F4 were prepared from asialo-fibrinogen by pronase digestion as described previously (Nemansky and Van den Eijnden, 1993). The peptide portion of GP-F2 consists for >90% of Gly-Glu-Asn and Glu-Asn in a ratio of 3:2; GP-F4 contains some additional amino acids. As-[alpha]1-AGP, as-fetuin, as-hCG[alpha], as-GP-F2, and as-GP-F4 were enzymatically degalacto-sylated with jack bean [beta]-galactosidase (0.2 U/µmol terminal Gal) in 50 mM sodium acetate pH 4.0 (10-20 mg of as-glycoprotein per ml) to yield the asialo/agalacto (as/ag) forms.

Bovine colostrum was obtained from a local farm. Polyclonal antiserum, raised against calf thymus [beta]4-GalT, was kindly donated by Dr. D.H.Joziasse (Vrije Universiteit Amsterdam, Amsterdam, The Netherlands).

UDP-[3H]GalNAc (8.7 Ci/mmol) and UDP [3H]Gal (10.6 Ci/mmol) were purchased from New England Nuclear, Boston, Massachusetts. The sugar nucleotide donors were diluted with unlabeled UDP-GalNAc and UDP-Gal (Sigma) respectively, to give the desired specific radioactivity. All other chemicals were obtained from commercial sources and were of the best quality available.

Preparation of bovine mammary gland membranes and purification of [beta]4-GalT from bovine colostrum

Bovine mammary gland (35 g), was washed in 0.15 M NaCl, cut in small pieces, and homogenized in 100 ml 0.25 M sucrose using a Polytron homogenizer (3 × 20 s at maximum speed). The homogenate (135 ml) was centrifuged for 10 min at 800 × g. The supernatant was then centrifuged for 60 min at 100,000 × g. Subsequently, the supernatant was discarded and the pellet was resuspended in 4 ml 0.25 M sucrose. All manipulations were carried out at 0-4°C. The protein concentration was determined according to Peterson (1977) using bovine serum albumin as a standard. Highly purified [beta]4-GalT was prepared from bovine colostrum as described previously (Blanken et al., 1982).

[beta]4-N-Acetylgalactosaminyltransferase ([beta]4-GalNAcT) assay

The standard incubation mixture contained 5 µmol sodium-cacodylate pH 7.0, 2 µmol MnCl2, 0.4 µl Triton X-100, 0.2 µmol ATP, 25 nmol UDP-[3H]GalNAc (1.5 Ci/mol), acceptor substrate (containing 50 nmol of terminal GlcNAc at the non-reducing end with the exception of compounds 16 and 17 which were present in an amount of 50 nmol), and bovine mammary gland microsomes (400 µg of protein) in a volume of 50 µl. Control incubations were carried out without acceptor substrates to correct for incorporation into endogenous acceptors. Where mentioned [alpha]-LA and polyclonal antiserum against [beta]4-GalT were added in the concentrations indicated. The mixtures were incubated for 60-150 min at 37°C. Incorporation of GalNAc into the acceptors was determined according to different methods depending on the type of acceptor as described below.

[beta]4-Galactosyltransferase ([beta]4-GalT) assay

The standard incubation mixture contained 5 µmol sodium cacodylate pH 7.2, 1 µmol MnCl2, 0.4 µl Triton X-100, 0.2 µmol ATP, 25 nmol UDP-[3H]Gal (1.0 Ci/mol), 50 nmol GlcNAc-S-pNP, and bovine mammary gland microsomes (400 µg of protein) in a volume of 50 µl. Controls, further additions, and incubation conditions were as described for the [beta]4-GalNAcT assay.

Assay method 1: oligosaccharide and glycopeptide acceptors

The reactions were terminated by the addition of 450 µl cold H2O. The samples were subsequently passed over a 0.5 ml Dowex 1-X8 (Cl- form) column. The column was washed three times with 0.5 ml H2O. The combined eluate and washes were assayed for radioactivity by liquid scintillation.

Assay method 2: oligosaccharide acceptors with hydrophobic aglycon

The reactions were stopped by addition of 950 µl 10 mM acetic acid. Incorporation of [3H]GalNAc or [3H]Gal was estimated by the use of Sep-Pak C-18 reverse-phase cartridges following the method of Palcic et al. (1988). The mixtures were applied to the Sep-Pak columns, which were washed with 30 ml of water. Radioactive products were eluted with 5 ml methanol. The eluate was evaporated to dryness, dissolved in 1 ml of water, and assayed for radioactivity.

Assay method 3: glycoprotein acceptors

Incubations with glycoprotein acceptors were stopped by adding 1 ml 0.1% bovine serum albumin and precipitation of the proteins with 1 ml 1 M HCl containing 2% (w/v) phosphotungstic acid. The precipitate was washed three times with 1 ml of the acid mixture and once with 1 ml methanol by resuspension, centrifugation and decantation. Finally, incorporation of radioactivity was estimated by solubilization of the precipitate in 200 µl Soluene (Packard) and the radioactivity was counted.

Assay method 4: as/ag-GP-F4 and as/ag-hCG[alpha] as acceptor substrates

Incubations with as/ag-GP-F4 and as/ag-hCG[alpha] were stopped by adding 150 µl cold 50 mM ammonium acetate (pH 5.2). The samples were passed over a Bio-Gel P-4 (200-400 mesh) column (0.7 × 18 cm), equilibrated and eluted in 50 mM ammonium acetate (pH 5.2). Fractions of 0.5 ml were collected and incorporation of [3H]GalNAc or [3H]Gal was calculated from the radioactivity in the fractions containing the peptide product.

Kinetic parameters

Kinetic parameters were estimated from Lineweaver-Burk plots that were obtained by varying the GlcNAc concentration from 2 to 50 mM and that of Glc from 10 to 500 mM and from 150 mM to 500 mM in the presence or absence of 12 mg per ml [alpha]-LA, respectively.

Large-scale incubations

A large-scale incubation with GlcNAc as acceptor substrate was conducted in order to isolate sufficient product for characterization by 400-MHz 1H-NMR spectroscopy. The incubation mixture contained 50 µmol sodium cacodylate pH 7.0, 20 µmol MnCl2, 2 µmol ATP, 2.5 µl Triton X-100, 0.75 µmol UDP-[3H]GalNAc (0.45 Ci/mol), 15.0 µmol GlcNAc, and bovine mammary gland membranes (31 mg of protein) in a volume of 0.5 ml. This mixture was incubated for 75 min at 37°C. Unused donor substrate was removed by passing the mixture over a 3.0 ml Dowex 1-X8 column (Cl--form). The combined run-through and three washes of 3 ml each were fractionated on a column (1.6 × 200 cm) of Bio-Gel P-4 (200-400 mesh) equilibrated and eluted with 50 mM ammonium acetate pH 5.2. Fractions were assayed for radioactivity, and fractions containing the radioactive oligosaccharide product were pooled and lyophilized.

The large-scale incubation with Glc as an acceptor, in the presence of [alpha]-LA, contained the following constituents: 50 µmol sodium cacodylate pH 7.0, 20 µmol MnCl2, 2 µmol ATP, 2.5 µl Triton X-100, 5 µmol GalNAc (to counteract hexosaminidase activity), 1.5 µmol UDP-[3H]GalNAc (0.23 Ci/mol), 0.25 mmol Glc, 6 mg [alpha]-LA, and bovine mammary gland membranes (34 mg of protein) in a volume of 0.5 ml. This mixture was incubated for 24 h at 37°C. The product was isolated by Dowex 1 chromatography and filtration on Bio-gel P-4 as described above. Fractions were assayed for radioactivity and analyzed by high-pH anion-exchange chromatography as described by Bakker et al. (1997).

400 MHz 1H-NMR spectroscopy

Samples were desalted on a column (1.0 × 42 cm) of Bio-Gel P-2 (200-400 mesh) run in water. They were treated three times with 2H2O (99.75 atom %; Merck, Darmstadt, Germany) at p2H 7 and room temperature with intermediate freeze-drying. Finally the sample was redissolved in 360 µl of 2H2O (99.95 atom %; Aldrich, Milwaukee, Wisconsin). 1H NMR spectroscopy was performed at 400 MHz on a Bruker MSL-400 spectrometer (facility of the Department of Physics, Vrije Universiteit, Amsterdam, The Netherlands) operating in the Fourier-transform mode. The probe temperature was kept at 300 K. Resolution enhancement of the spectra was achieved by Lorentzian-to-Gaussian transformation. Chemical shifts are expressed downfield from internal 4,4-dimethyl-4-silapentane-1-sulfonate, but were actually measured by reference to internal acetone ([delta] = 2.225 ppm).

Acknowledgments

We are indebted to Drs. L.Anderson, J.Lönngren, O.Hindsgaul, G.Strecker, A.Veyrières, M.Haraldsson, R.A.Wijnands, and D.H.Joziasse for their kind gifts of oligosaccharides, glycoproteins, or antiserum. We thank Mrs. C.A.M.Koeleman for recording the 400 MHz 1H-NMR spectra.

Abbreviations

[alpha]1-AGP, [alpha]1-acid glycoprotein; [alpha]-LA, [alpha]-lactalbumin; (as/ag)-GP-F2, (desialylated and degalactosylated) diantennary glycopeptide of fibrinogen; [beta]4-GalT, UDP-Gal:GlcNAc[beta]-R [beta]1->4-galactosyltransferase; [beta]4-GalNAcT, UDP-GalNAc:GlcNAc[beta]-R [beta]1->4-N-acetylgalactosaminyltransferase; Gal, galactose; GalNAc, N-acetylgalactosamine, Glc, glucose; GlcNAc, N-acetylglucosamine; hCG[alpha], [alpha]-subunit of human chorionic gonadotrophin; lacdiNAc, N,N[prime]-diacetyllactosediamine; lacNAc, N-acetyllactosamine; pNP, para-nitrophenol.

References

Almeida ,R., Amado,M., David,L., Levery,S.B., Holmes,E.H., Merkx,G., Van Kessel,A.G., Rygaard,E., Hassan,H., Bennett,E. and Clausen,H. (1997) A family of human [beta]-4-galactosyltransferases - Cloning and expression of two novel UDP-galactose:[beta]-N-acetylglucosamine [beta]-1,4-galactosyltransferases, [beta]4Gal-T2 and [beta]4Gal-T3. J. Biol. Chem., 272, 31979-31991. MEDLINE Abstract

Aoki ,N., Furukawa,K., Iwatsuki,K., Noda,A., Sato,T., Nakamura,R. and Matsuda,T. (1995) A bovine IgG heavy chain contains N-acetylgalactosaminylated N-linked sugar chains. Biochem. Biophys. Res. Commun., 210, 275-280. MEDLINE Abstract

Baenziger ,J.U. and Green,E.D. (1988) Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim. Biophys. Acta, 947, 287-306. MEDLINE Abstract

Bakker ,H., Agterberg,M., Van Tetering,A., Koeleman,C.A.M., Van den Eijnden,D.H. and Van Die,I. (1994) A Lymnaea stagnalis gene, with sequence similarity to that of mammalian [beta]1->4-galactosyltransferases, encodes a novel UDP-GlcNAc:GlcNAc[beta]-R [beta]1->4-N-acetylglucosaminyltransferase. J. Biol. Chem., 269, 30326-30333. MEDLINE Abstract

Bakker ,H., Schoenmakers,P.S., Koeleman,C.A.M., Joziasse,D.H., Van Die,I. and Van Den Eijnden,D.H. (1997) The substrate specificity of the snail Lymnaea stagnalis UDP-GlcNAc:GlcNAc[beta]-R [beta]4-N-acetylglucosaminyltransferase reveals a novel variant pathway of complex-type oligosaccharide synthesis. Glycobiology, 7, 539-548. MEDLINE Abstract

Bergwerff ,A.A., Van Oostrum,J., Kamerling,J.P. and Vliegenthart,J.F.G. (1995) The major N-linked carbohydrate chains from human urokinase-the occurrence of 4-O-sulfated, ([alpha]2-6)-sialylated or ([alpha]1-3)-fucosylated N-acetylgalactosamine ([beta]1-4)-N-acetylglucosamine elements. Eur. J. Biochem., 228, 1009-1019. MEDLINE Abstract

Blanken ,W.M., Hooghwinkel,G.J.M. and Van den Eijnden,D.H. (1982) Biosynthesis of blood group I and i substances. Specificity of bovine colostrum [beta]-N-acetyl-d-glucosaminide [beta]1->4-galactosyltransferase. Eur. J. Biochem., 127, 547-552. MEDLINE Abstract

Blanken ,W.M., Van Vliet,A. and Van den Eijnden,D.H. (1984) Branch specificity of bovine colostrum and calf thymus UDP-Gal:N-acetylglucosaminide [beta]1->4-galactosyltransferase. J. Biol. Chem., 259, 15131-15135. MEDLINE Abstract

Coddeville ,B., Strecker,G., Wieruszeski,J.M., Vliegenthart,J.F.G., van Halbeek,H., Peter-Katalinic,J., Egge,H. and Spik,G. (1992) Heterogeneity of bovine lactotransferrin glycans. Characterization of [alpha]-d-Galp-(1->3)-[beta]-d-Gal- and [alpha]-NeuAc-(2->6)-[beta]-d-GalpNAc-(1->4)-[beta]-d-GlcNAc-substituted N-linked glycans. Carbohydr. Res., 236, 145-164. MEDLINE Abstract

Colman ,A. (1996) Production of proteins in the milk of transgenic livestock: problems, solutions, and successes. Am. J. Clin. Nutr., 63, 639-645.

Dell ,A., Morris,H.R., Easton,R.L., Panico,M., Patankar,M., Oehninger,S., Koistinen,R., Koistinen,H., Seppala,M. and Clark,G.F. (1995) Structural analysis of the oligosaccharides derived from glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities. J. Biol. Chem., 270, 24116-24126. MEDLINE Abstract

Dharmesh ,S.M., Skelton,T.P. and Baenziger,J.U. (1993) Co-ordinate and restricted expression of the ProXaaArg/Lys-specific GalNAc-transferase and the GalNAc[beta]1,4GlcNAc[beta]1,2Man[alpha]-4-sulfotransferase. J. Biol. Chem., 268, 17096-17102. MEDLINE Abstract

Do ,K.Y., Do,S.I. and Cummings,R.D. (1995) [alpha]-Lactalbumin induces bovine milk [beta]1,4-galactosyltransferase to utilize UDP-GalNAc. J. Biol. Chem., 270, 18447-18451. MEDLINE Abstract

Fiete ,D., Srivastava,V., Hindsgaul,O. and Baenziger,J.U. (1991) A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc[beta]1,4GlcNAc[beta]1,2Man[alpha] that mediates rapid clearance of lutropin. Cell, 67, 1103-1110. MEDLINE Abstract

Girardet ,J.M., Coddeville,B., Plancke,Y., Strecker,G., Campagna,S., Spik,G. and Linden,G. (1995) Structure of glycopeptides isolated from bovine milk component PP3. Eur. J. Biochem., 234, 939-946. MEDLINE Abstract

Green ,E.D. and Baenziger,J.U. (1988) Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem., 263, 25-35. MEDLINE Abstract

Grinnell ,B.W., Hermann,R.B. and Yan,S.B. (1994) Human protein C inhibits selectin-mediated cell adhesion: role of unique fucosylated oligosaccharide. Glycobiology, 4, 221-225. MEDLINE Abstract

Hao ,Y.-L. and Wickerhauser,M. (1973) Development of large-scale fractionation methods. IV. A simple method for the purification of [alpha]1-acid glycoprotein. Biochim. Biophys. Acta, 322, 99-108. MEDLINE Abstract

Hård ,K., Van Zadelhoff,G., Moonen,P., Kamerling,J.P. and Vliegenthart,J.F.G. (1992) The Asn-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male. Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains. Eur. J. Biochem., 209, 895-915.

Hathaway ,H.J., Runyan,R.B., Khounlo,S. and Shur,B.D. (1991) Purification and characterization of avian [beta]1,4-galactosyltransferase: comparison with the mammalian enzyme. Glycobiology, 1, 211-221. MEDLINE Abstract

Hill ,R.L. and Brew,K. (1975) Lactose synthetase. Adv. Enzymol. Rel. Areas Mol. Biol., 43, 411-490.

Hiyama ,J., Weisshaar,G. and Renwick,A.G.C. (1992) The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin. Glycobiology, 2, 401-409. MEDLINE Abstract

Hooper ,L.V., Beranek,M.C., Manzella,S.M. and Baenziger,J.U. (1995) Differential expression of GalNAc-4-sulfotransferase and GalNAc-transferase results in distinct glycoforms of carbonic anhydrase VI in parotid and submaxillary glands. J. Biol. Chem., 270, 5985-5993. MEDLINE Abstract

Houdebine ,L.M. (1995) The production of pharmaceutical proteins from the milk of transgenic animals. Repr. Nutr. Dev., 35, 609-617.

Maga ,E.A. and Murray,J.D. (1995) Mammary gland expression of transgenes and the potential for altering the properties of milk. Biotechnology, 13, 1452-1457.

Manzella ,S.M., Hooper,L.V. and Baenziger,J.U. (1996) Oligosaccharides containing [beta]-1,4-linked N-acetylgalactosamine, a paradigm for protein-specific glycosylation. J. Biol. Chem., 271, 12117-12120. MEDLINE Abstract

Masri ,K.A., Appert,H.E. and Fukuda,M.N. (1988) Identification of the full-length coding sequence for human galactosyltransferase ([beta]-N-acetylglucosaminide [beta]1,4-galactosyltransferase). Biochem. Biophys. Res. Commun., 157, 657-663. MEDLINE Abstract

Mengeling ,B.J., Manzella,S.M. and Baenziger,J.U. (1995) A cluster of basic amino acids within an [alpha]-helix is essential for [alpha]-subunit recognition by the glycoprotein hormone N-acetylgalactosaminyltransferase. Proc. Natl. Acad. Sci. USA, 92, 502-506. MEDLINE Abstract

Messer ,M. and Nicholas,K.R. (1991) Biosynthesis of marsupial milk oligosaccharides: characterization and developmental changes of two galactosyltransferases in lactating mammary glands of the tammar wallaby, Macropus eugenii. Biochim. Biophys. Acta, 1077, 79-85. MEDLINE Abstract

Mulder ,H., Spronk,B.A., Schachter,H., Neeleman,A.P., Van den Eijnden,D.H., De Jong-Brink,M., Kamerling,J.P. and Vliegenthart,J.F.G. (1995) Identification of a novel UDP-GalNAc:GlcNAc[beta]-R [beta]1-4-N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis. Eur. J. Biochem., 227, 175-185. MEDLINE Abstract

Nakata ,N., Furukawa,K., Greenwalt,D.E., Sato,T. and Kobata,A. (1993) Structural study of the sugar chains of CD36 purified from bovine mammary epithelial cells-occurrence of novel hybrid-type sugar chains containing the Neu5Ac[alpha]2->6GalNAc[beta]1->4GlcNAc and the Man[alpha]1->2Man[alpha]1->3Man[alpha]1->6Man groups. Biochemistry, 32, 4369-4383. MEDLINE Abstract

Narasimhan ,S., Freed,J.C. and Schachter,H. (1985) Control of glycoprotein synthesis. Bovine milk UDPgalactose:N-acetylglucosamine [beta]-4-galactosyltransferase catalyses the preferential transfer of galactose to the GlcNAc[beta]1,2Man[alpha]1,3- branch of both bisected and nonbisected complex biantennary asparagine-linked oligosaccharides. Biochemistry, 24, 1694-1700. MEDLINE Abstract

Neeleman ,A.P. and Van den Eijnden,D.H. (1996) [alpha]-Lactalbumine affects the acceptor specificity of Lymnaea stagnalis albumen gland UDP-GalNAc:GlcNAc[beta]-R [beta]1->4-N-acetylgalactosaminyltransferase. Proc. Natl. Acad. Sci. USA, 93, 10111-10116. MEDLINE Abstract

Neeleman ,A.P., Van der Knaap,W.P.W. and Van den Eijnden,D.H. (1994) Identification and characterization of a UDP-GalNAc:GlcNAc[beta]-R [beta]1->4-N-acetylgalactosaminyltransferase from cercariae of the schistosome Trichobilharzia ocellata. Catalysis of a key step in the synthesis of N,N[prime]- diacetyllactosediamino (lacdiNAc)-type glycans. Glycobiology, 4, 641-651. MEDLINE Abstract

Nemansky ,M. and Van den Eijnden,D.H. (1992) Bovine colostrum CMP-NeuAc:Gal[beta](1->4)GlcNAc-R [alpha](2->6)-sialyltransferase is involved in the synthesis of the terminal NeuAc[alpha](2->6)GalNAc[beta](1->4)GlcNAc sequence occurring on N-linked glycans of bovine milk glycoproteins. Biochem. J., 287, 311-316. MEDLINE Abstract

Nemansky ,M. and Van den Eijnden,D.H. (1993) Enzymatic characterization of CMP-NeuAc:Gal[beta]1->4GlcNAc-R [alpha](2->3)-sialyltransferase from human placenta. Glycoconjugate J., 10, 99-108.

Palcic ,M.M. and Hindsgaul,O. (1991) Flexibility in the donor substrate specificity of [beta]1,4-galactosyltransferase: application in the synthesis of complex carbohydrates. Glycobiology, 1, 205-209. MEDLINE Abstract

Palcic ,M.M., Heerze,L.D., Pierce,M. and Hindsgaul,O. (1988) The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays. Glycoconjugate J., 5, 49-63.

Peterson ,G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem., 83, 346-356. MEDLINE Abstract

Pohlentz ,G., Klein,D., Schwarzmann,G., Schmitz,D. and Sandhoff,K. (1988) Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver. Proc. Natl. Acad. Sci. USA, 85, 7044-7048. MEDLINE Abstract

Powell ,J.T. and Brew,K. (1974) Glycosyltransferases in the golgi membranes of onion stem. Biochem. J., 142, 203-209. MEDLINE Abstract

Rivera-Marrero ,C.A. and Cummings,R.D. (1990) Schistosoma mansoni contains a galactosyltransferase activity distinct from that typically found in mammalian cells. Mol. Biochem. Parasitol., 43, 59-68. MEDLINE Abstract

Rohrmann ,K., Niemann,R. and Buddecke,E. (1985) Two N-acetylgalactosaminyltransferase are involved in the biosynthesis of chondroitin sulfate. Eur. J. Biochem., 148, 463-469. MEDLINE Abstract

Rosen ,J.M., Li,S., Raught,B. and Hadsell,D. (1996) The mammary gland as a bioreactor: factors regulating the efficient expression of milk protein-based transgenes. Am. J. Clin. Nutr., 63, 627-632.

Sato ,T., Furukawa,K., Bakker,H., Van den Eijnden,D.H. and Van Die,I. (1998) Molecular cloning of a human cDNA encoding a novel [beta]4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc [beta]1,4-galactosyltransferase. Proc. Natl. Acad. Sci. USA, 95, 472-477. MEDLINE Abstract

Sato ,T., Furukawa,K., Greenwalt,D.E. and Kobata,A. (1993) Most bovine milk fat globule membrane glycoproteins contain asparagine-linked sugar chains with GalNAc[beta]1->4GlcNAc groups. J. Biochem., 114, 890-900. MEDLINE Abstract

Sato ,T., Takio,K., Kobata,A., Greenwalt,D.E. and Furukawa,K. (1995) Site-specific glycosylation of bovine butyrophilin. J. Biochem., 117, 147-157. MEDLINE Abstract

Schachter ,H. and Roseman, S. (1980) Mammalian glycosyltransferases. Their role in the synthesis and function of complex carbohydrates and glycolipids. In Lennarz,W.J. (ed.), The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, New York, pp. 85-160.

Schanbacher ,F.L. and Ebner,K.E. (1970) Galactosyltransferase acceptor specificity of the lactose synthetase A protein. J. Biol. Chem., 245, 5057-5061. MEDLINE Abstract

Shaper ,N.L., Meurer,J.A., Joziasse,D.H., Chou,T.D.D., Smith,E.J., Schnaar,R.L. and Shaper,J.H. (1997) The chicken genome contains two functional nonallelic [beta]-1,4-galactosyltransferase genes-chromosomal assignment to syntenic regions tracks fate of the two gene lineages in the human genome. J. Biol. Chem., 272, 31389-31399. MEDLINE Abstract

Sheares ,B.T. and Carlson,D.M. (1984) Two distinct UDP-galactose:2-acetamido-2-deoxy-d-glucose 4[beta]-galactosyltransferases in porcine trachea. J. Biol. Chem., 259, 8045-8047. MEDLINE Abstract

Siciliano ,R.A., Morris,H.R., McDowell,R.A., Azadi,P., Rogers,M.E., Bennett,H.P. and Dell,A. (1993) The Lewis x epitope is a major non-reducing structure in the sulphated N-glycans attached to Asn-65 of bovine pro-opiomelanocortin. Glycobiology, 3, 225-239. MEDLINE Abstract

Smith ,P.L. and Baenziger,J.U. (1988) A pituitary N-acetylgalactosamine transferase that specifically recognizes glycoprotein hormones. Science, 242, 930-932. MEDLINE Abstract

Smith ,P.L. and Baenziger,J.U. (1992) Molecular basis of recognition by the glycoprotein hormone-specific N-acetylgalactosamine-transferase. Proc. Natl. Acad. Sci. USA, 89, 329-333. MEDLINE Abstract

Smith ,P.L., Bousfield,G.R., Kumar,S., Fiete,D. and Baenziger,J.U. (1993) Equine lutropin and chorionic gonadotropin bear oligosaccharides terminating with SO4-4-GalNAc and Sia[alpha]2,3Gal, respectively. J. Biol. Chem., 268, 795-802. MEDLINE Abstract

Soh ,C.P., Donald,A.S., Feeney,J., Morgan,W.T. and Watkins,W.M. (1989) Enzymic synthesis, chemical characterisation and Sda activity of GalNAc[beta]1-4[NeuAc[alpha]2-3]Gal[beta]1-4GlcNAc and GalNAc[beta]1-4[NeuAc[alpha]2-3]Gal[beta]1-4Glc. Glycoconjugate J., 6, 319-332.

Srivatsan ,J., Smith,D.F. and Cummings,R.D. (1994) Demonstration of a novel UDP-GalNAc:GlcNAc [beta]1-4-N-acetylgalactosaminyltransferase in extracts of Schistosoma mansoni. J. Parasitol., 80, 884-890. MEDLINE Abstract

Taguchi ,T., Kitajima,K., Muto,Y., Inoue,S., Khoo,K.H., Morris,H.R., Dell,A., Wallace,R.A., Selman,K. and Inoue,Y. (1995) A precise structural analysis of a fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of euryhaline killi fish (Fundulus heteroclitus)-novel penta-antennary N-glycan chains with a bisecting N-acetylglucosaminyl residue. Glycobiology, 5, 611-624. MEDLINE Abstract

Tilley , C.A., Singer, A., Harris-Brandts, M. and Moscarello, M.A. (1991) The major oligosaccharide of bovine [alpha]-lactalbumin carries terminal [beta]-linked GalNAc. Glycoconjugate J., 8, 249-250.

Tomiya ,N., Awaya,J., Kurono,M., Hanzawa,H., Shimada,I., Arata,Y., Yoshida,T. and Takahashi,N. (1993) Structural elucidation of a variety of GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase. J. Biol. Chem., 268, 113-126. MEDLINE Abstract

Van den Eijnden ,D.H., Bakker,H., Neeleman,A.P., Van den Nieuwenhof,I.M. and Van Die,I. (1997) Novel pathways in complex-type oligosaccharide synthesis. New vistas opened by studies in invertebrates. Biochem. Soc. Trans., 25, 887-893. MEDLINE Abstract

Van Die ,I., Van Tetering,A., Bakker,H., Van den Eijnden,D.H. and Joziasse,D.H. (1996) Glycosylation in lepidopteran insect cells: identification of a [beta]1->4-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains. Glycobiology, 6, 157-164. MEDLINE Abstract

Van Die ,I., Bakker,H. and Van den Eijnden,D.H. (1997) Identification of conserved amino acid motifs in members of the [beta]1->4-galactosyltransferase gene family. Glycobiology, 7, v-viii. MEDLINE Abstract

Weisshaar ,G., Hiyama,J. and Renwick,A.G.C. (1990) Site specific N-glycosylation of ovine lutropin. Eur. J. Biochem., 192, 741-751. MEDLINE Abstract

Weisshaar ,G., Hiyama,J., Renwick,A.G. and Nimtz,M. (1991) NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin. Eur. J. Biochem., 195, 257-268. MEDLINE Abstract

Yan ,S.B., Chao,Y.B. and Van Halbeek,H. (1993) Novel Asn-linked oligosaccharides terminating in GalNAc[beta](1->4)[Fuc[alpha](1->3)]GlcNAc[beta](1->[bull]) are present in recombinant human protein C expressed in human kidney 293 cells. Glycobiology, 3, 597-608. MEDLINE Abstract

Young ,M.W., Okita,W.B., Brown,E.M. and Curling,J.M. (1997) Production of biopharmaceutical proteins in the milk of transgenic dairy animals. Biopharmacy, 10, 34-38.


1To whom correspondence should be addressed


This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 6 Feb 1999
Copyright©Oxford University Press, 1999.