Interactions of cell-surface galactosyltransferase with immunoglobulins

0161-5890193$6.00 i- 0.00 @ 1993 Pergamon Press LM

Molecular hmmo~ogy, Vol. 30, No. 3, pp. 265-275, 1993

Printed in Great Britain.

INTERACTIONS OF CELL-SURFACE GALACTOSYLTRANSFERASE WITH IMMUNOGLOBULINS MILAN TOMANA,*~ Jwr -N,*

ZINA ~oL~~~,~ ROSE KUJXAVY,~ J. CLAUDE BENNETT*$ and JIRI MES’MXKY*$

*Division of Clinical Immunolo~ and Rheumatolo~, Department of Medicine and SDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. (First received 7 August 1991; accepted in revised form 4 August 1992) ~a~-petition of the activity of ~-1,~galactosyltransfera~ @=1,4=GT) in s~~nsions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinema ceil line HT-29, the monoeyte-iike cell line U937, and human splenic B and T lymphocytes suggested the presence of /I-l,CGT, in an enzymatically active form, on plasma membranes. The presence of B=l,CGT on eel1 surfaces was also indicated from the effect of t~psinization of live cells, which significantly reduced cell surface p=l,CGT activity, but did not a&et the activity associated with cytoplasmic membranes. Furthermore, the presence of b=l,CGT on the cell surface was demonstrated by indirect immunofluoreseenee staining of cells with anti-b-1,4-GT antibody. The detection of radioactivity in i~unoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn2+ and UDP-[3H~=gaiacto~, indicated that Ig molecules were galactosylated. In the absence of UDP-[3H]=gabetose, ~=1,4=GT on eel1 surfaces, or immobilized on Sepharose=4B, formed stable complexes with galactose acceptors, including Ig. The efhciency of binding decreased in the order: J chain > a chain > p chain > polymeric IgA2 > monomeric/polymeric IgAl > IgM B IgG. Thus, /I-1,4-CT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the j=l,4=GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled hgand binding assays. The binding was inhibitable by EDTA, cr-lactalbumin (in the presence of glucose), GlcNAc, or uridine ~,5‘dialdehyde. At 37°C the apparent affinity eon&ants and association rate constants of interaction between eel1 surface fi=1,4=GT on ~utar~dehyde-fix~ HT-29 and U937 cells and ar2 chain or monomeric IgAl were in the range from 7.1 x 10’ to 4.6 x 108M-l and from 1 x 10s to 3 x lo6 M-’ s-l, respectively. The dissociation rate constants and half time of dissociation caiculated from these data were in the range from 2.1 x IO-’ to 5.0 x IO-* s-i and from 33 to 1380 s, respectively. The number of a2 or IgAl molecules bound per HT-29 and UP37 cell were in the range from 1.9 x 10” to 1.3 x 106. The binding of IgA by the cell surface /I-1,4=GT was not associated with internalization or the catabolic degradation of the ligand.

Galactosyltransferases are a family of enzymes, which catalyze the transfer of galactose (Gal) from UDP-Gal to appropriate acceptors. These enzymes are highly specific with respect to the type of linkage formed and the nature of the acceptor. The most widely dist~boted

is /I-1,4=galactosyltransferase @=1,4=GT) which catalyzes the transfer of Gal to ~=a~tyl~ucosamine (GlcNAc) either free or as part of a glycoconjugate (for review see Schachter and Roseman, 1980). The galactosyltransferase activity is usually associated with intracellular membranes, mainly of the Golgi complex. However, it has also been demonstrated on plasma membranes (Roth et al., 1971; Shur and Roth, 1975; Me&t et ai,, 1977; Pierce et al., 1980) and in soluble form in biological fluids such as amniotic (Nelson et al.,

tAuthor to whom correspondence should be addressed: Milan Tornana, University of Alabama at Birmingham, CHSB 19, UAB Station, Bi~in~~~ Alabama 35294, U.S.A.

1973) and eerebrospinai (Ko et al., 1973) fluids, serum (Kim et al., 1972), milk (Babad and Hassid, 1964, Brodbeck and Ebner, 1966), tears and saliva (Tomana et al., 1993) and malignant effusions (Wilson et al., 1983). Galactosyltransferase may play a role in malignancy (Podolsky and Weiser, 1975; Chatterjee and Kim, 1977; Kessel et af., 1977; Podolsky et al., 1981; Weiser and Wilson, 1981) and cell surface phenomena involving cell-cell and cell-matrix interactions including, fertilization (Roth and White, 1972; Shur 1982a, 1989; Lopez et al., 1985), mesenchymal cell migration (Runyan et al., 1986), intercellular adhesion (Roseman, 1971), morphogenesis and homeostasis (Ram and Munjal, 1985), and cell spreading on laminin (Runyan et al., 1988). It has also been demonstrated that galactosyltransferase on the surface of embryonal carcinoma cells is capable of gal~tosylating exogenous acceptors as well as those expressed on the cell membrane (Shur, 19823). Recent studies have shown that galactosyltransferase from human milk isolated by affinity chromatography on immobilized human IgM reacted specifically with human milk &A. (McGuire er al., 1988, 1989). In the 265

266

M. TOMANAef

preceding paper (Tomana et al., 1993), we confirmed and extended this observation by showing that in the absence of UDP-[3H]-Gal, fl-1,4-GT from human colostrum forms complexes with a number of acceptors, including Ig of all major isotypes and some of their component chains, and that these complexes are present not only in milk, but also in serum. It is well documented that IgA, which is present in various molecular forms in serum and external secretions, interacts with a broad spectrum of mo~hologically and functionally diverse cells, including monocytes, macrophages, polymorphonuclear leukocytes, eosinophils, natural killer cells, B and T lymphocytes, hepatocytes and epithelial cells covering mucosal surfaces (for reviews see Mestecky and McGhee, 1987; Mestecky et al., 1988). Such interactions are of functional importance, as the IgA bound to various cell types may be internalized and selectivity transported into external secretions, or internalized and subsequently catabolized, or may stimulate the release of biologically active substances. At least three IgA-specific receptors have been identified on mammalian cells: secretory component (SC), also called polyimmunoglobulin receptor (Mostov et al., 1984; Brandtzaeg, 1985; Mestecky and McGhee, 1987; Mestecky et al., 1988), which is present on epithelial cells and hepatocytes of rodents and lagomorphs; asialoglycoprotein receptor (ASGPR), which was identified on human and rodent hepatocytes (Stockert et al., 1982; Tomana et al., 1988~); and a highly glycosylated receptor with molar mass 60 kD, expressed on monocytes (Monteiro et al., 1990; Maliszewski et al., 1990) and polymorphonuclear leukocytes (Fanger et al., 1980). SC is essential in the selective transport of polymeric IgA into external secretions (Brandtzaeg, 1985), while ASGPR is responsible for the internalization and catabolism of all molecular forms of IgA (Moldoveanu et al., 1988; Tomana et al., 1988a). The function of the third receptor remains enigmatic. The aim of the present study was to examine cells known to bind IgA with respect of their expression of plasma membrane p-1,4GT and the potential of the p-1,4-GT to serve as an IgA receptor. SERIALS

AND ~THODS

Cells

Cell suspensions from mouse liver were obtained by in situ perfusion of liver with collagenase as described by Renton et al. (1978) and modified by Phillips et al. (1988). Parenchymal and nonparench~al cells were separated by repeated centrifu~ation at 50 g for 5 min in Joklik’s tissue culture medium (Flow Laboratories, Inc., McLean, VA) containing 5% heat-inactivated fetal calf serum (FCS) (ICN Flo, Los Angeles, CA). Parenchymal cells were then passed through Nitex (75 pm mesh; Tetko, Inc., Ehnsford, NY) to remove clumps of cells and tissue debris. The purified parenchymal cell preparation contained 80-95% hepatocytes with viability ranging from 78-92%, as assessed by Trypan Blue exclusion. The human hepatoma cell line, Hep G2, the

at.

colonic epithelial adenocarcinoma cell line, HT-29, and the macrophage-like cell line, U937, were grown in RPM1 1640 medium containing glutamine, heat-inactivated 10% FCS, 50 I.U. of ~nicillin~ and 50 mg/ml streptomycin (ICN Flow). The cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Human splenic B and T lymphocytes were prepared from a tissue obtained after splenectomy. Single cell suspension was obtained by mechanical dissociation using 60 mesh wire screen, and the mononuclear cell fraction was collected after Ficoll-Hypaque gradient centrifugation. Purified B and T lymphocytes were obtained by rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells and separating the non-rosetted from rosetted cells by FicollHypaque gradient ~ntrifugation (Saxon et al., 1976). Lymphocytes were maintained in RPM1 1640 medium with glutamine, FCS and antibiotics. Non-viable cells were removed by centrifugation through LSM solution (Ficoll-diatrizoate salt, Organon Teknika Corporation, Durham, NC) (DeVries et al., 1973). Cell count and viability were determined by Trypan Blue exclusion. Cells were fixed with glutaraldehyde according to Williams (1973). The cell suspension was incubated for 5 min at room temp with an equal volume of a 0.25% solution of glutaraldehyde (Type II, Sigma Chemical Company, St Louis, MO) in Puck saline (Gibco Laboratories, Grand Island, NY). The reaction was terminated by addition of 5% (w/v) bovine serum albumin (BSA) in Puck saline. Preparation and radioiodination of proteins Ig, their component chains, and fi-1,4-GT were prepared as described in the preceding paper (Tomana et al., 1993). Proteins were radiolabeled with carrier-free Na’2SI (New England Nuclear, Boston, MA) by the lactoperoxidase method (Marchalonis, 1969). At least 96% of the radioactivity was precipitable with 10% trichloracetic acid. Proteins not used immediately were aliquoted and stored at -70°C. Assays of fl-1,4-GT activity p-1,4-GT activity in suspensions of viable cells was assayed as described (Shur, 1982b), with a few modifications. Cells were washed three times by centrifugation in medium B (20 mM 2-[N-morpholinol-ethanesulfonic acid [MES], pH 7.2, 140 mM NaCl, and 5 mM KCl), and resuspended in 100 ,u1of the same medium supplemented with 10 mM MnCl,, 13.4pM UDP-[3H]-Gal (New England Nuclear, Boston, MA) (74.6 Ci/mol), 20 mM GlcNAc or 20 mM glucose and 1 mg/ml ff-la~talbumin. Assay mixtures were incubated for 1 hr at 37”C, after which the reaction was terminated with 10 ~1 of ice-cold 0.25 M EDTA, buffered with Tris, pH 7.2. Cells were pelleted by centrifugation and an aliquot of the supernatant was subjected to ion-exchange chromatography on a column of AGl -X8 ( 100-200 mesh in formate form; Bio-Rad Laboratories, Richmond, CA) to separate the galactosylated product from UDP-[3H]-Gal. Parallel assays were carried out without exogenous acceptor, to

267

Cell-surface galactosyltransferase determine activity to endogenous acceptors on the cell surface. When a glycoprotein was used as an exogenous acceptor, the reaction was terminated by the addition of 1 ml 5% (w/v) phosphotungstic acid in 2 M HCl to an aliquot of the supernatant. The precipitated protein was washed with 1% phosphotungstic acid in 0.5 M HCl. The reaction product was solubilized in ACS scintillation cocktail and the radioactivity counted in a Searle liquid scintillation spectrometer, as previously described (Tomana et al., 1992). Binding of glycoproteins to cell surface /I-1,4-GT

Viable cells were washed with Medium B and incubated with radioiodinated glycoproteins in the same medium, supplemented with 10 mM MnCl,. After incubation at 37°C for 60 min, the reaction was terminated by the addition of an excess of ice-cold Medium B, containing Mn2+. Cells were then pelleted by centrifugation, washed, and their radioactivity determined in a y spectrometer (model B5110 Packard Instrument Co., Inc., Downers Grove, IL). The binding of radiolabeled ligands to glutaraldehyde-fixed cells was carried out in 25 mM sodium cacodylate, pH 7.2, containing 17-25 mM MnCl,. The reaction was terminated by separating the cells from the medium by centrifugation through oil (Nilsson and Berg, 1977; Baenziger and Fiete, 1980). Aliquots of cell suspensions were layered on 100 ~1 of a mixture of dibutyl phthalate and dinonyl phthalate (7 : 3) in a 250 ~1 plastic test tube. Following centrifugation for 3 min at 12,OOOg,the water layer was frozen in dry ice-ethanol and the test tube was cut in the middle of the oil layer. The radioactivity of the bottom part, containing cells and the upper part containing supernatant, was measured in a y spectrometer. The non-specific binding was estimated by incubating cells in a medium that contained an inhibitor of /I-1,4-GT binding activity (EDTA) or, by adding radiolabeled ligands to cells which had been previously incubated at 58°C for 30min to inhibit their fl-1,4-GT activity. Immunofuorescence

microscopy

Viable HT-29 and Hep G2 cells washed with, and suspended in, PBS-0.1% NaN, were incubated for 45 min at 0°C with heat-inactivated rabbit antiserum to human /I- 1,4-GT, diluted 1: 50. Following additional washing, cells were incubated with tetramethylrhodamine isothiocyanate (TRITC)-goat anti-rabbit IgG (Southern Biotechnology, Birmingham, AL). Controls were incubated with secondary antibody only. To demonstrate interactions of Ig with cell surface /I-1,4GT, viable HT-29 and Hep G2 cells were washed with and suspended in Medium B supplemented with 10 mM MnCl, and 1% BSA and incubated for 1 hr at 0°C with biotinylated a2 and J chains (both isolated from plgA2 and labeled with biotin as described [Jackson et al., 19821). After additional washing, cells were incubated with avidin-aminomethyl coumarin acetate (AMCA; Jackson ImmunoResearch Laboratories Inc., Avondale, PA). Control cells were incubated with AMCA only. The staining procedures were carried out in the above Mn*+-

containing medium at 0°C. The stained cells were examined with a Leitz Orthoplan immunofluorescence microscope (E. Leitz Inc., Rockleigh, NJ) equipped with epi-illumination and a set of excitation and barrier filters for red and blue fluorescence. RESULTS Cell surface /3- 1,4-GT activity

b-1,4-GT activity toward exogenous GlcNAc and galactosyltransferase activity toward endogenous aceeptors present on the cell surface was detected in suspensions of viable mouse hepatocytes, the human monocyte-like cell line U937, the colonic epithelial adenocarcinoma cell line HT-29, the hepatoma cell line Hep G2, and splenic B and T lymphocytes, which were incubated in a medium containing MnCl, and UDP[3H]-Gal (Table 1). Because the cell viability was relatively high (8499%), the /I-1,4-GT activity detected was not likely to be the result of dead or lysed cells that released their intracellular transferase into the incubation medium. The presence of enzymatically active /3-l ,4-GT on the cell surface was also indicated by trypsinization experiments in which treatment with trypsin-EDTA reduced the /I-1,4-GT activity in suspensions of HT-29 cells by more than 75%, without affecting the cell viability. Substantially higher fl-1,4-GT activity was detected when trypsin-treated cells were subsequently lysed with Triton X-100, which released the b - 1,4-GT associated with the cytoplasmic membranes (Table 2). The presence of fi-1,4-GT on plasma membranes was further demonstrated by immunofluorescence using living cells, rabbit antibody to human /I-1,4-GT and TRITC-conjugated goat anti-rabbit IgG (Fig. 1). The possibility that the /I-1,4-GT activity was the result of shedding of the enzyme into the assay medium was examined. Hep G2 cells (96% viable) were incubated for 1 hr at 37°C in a medium lacking UDPGal. Supernatants (from 5 x 10’ cells) were then collected and assayed for /I-1,4-GT activity toward Table 1. Galactosyltransferase activity in suspensions of live cells” Acceptor Endogenous b GlcNAc c Cells Mouse hepatocytes HT-29 Hep G2 u937 B lymphocytesd T lymphocytesd

Viability W) 86.2 99.0 96.7 97.3 97.1 93.4

@moles product/ 1 x lo5 cells/h) 5.91 5.15 4.32 2.66 3.26 2.96

f + + f f +

0.95 0.65 0.69 0.38 0.55 0.51

17.38 f 3.30 13.63 f 2.21 9.19 & 2.43 6.85 f 1.32 10.62 f 2.95 6.81 * 1.44

“Assays are the mean f SD of four determinations. bActivities toward endogenous acceptors were corrected for background detected in suspensions of cells in which GT activity was inhibited by incubation at 58°C for 30min. ‘The activity toward GlcNAc was corrected for background and for activity toward endogenous acceptors. ‘% and T lymphocytes were isolated from human spleen.

26X

M. TWANA et cd. Table 2. Surface

and cytoplasmic

,8-1,4-GT

activity

of HT-29 cells”

/j-1,4-GT activity” (pmoles product’ 3.1 x 10’ cells;‘hr) Intact cells Intact cells + trypsin’ Intact cells + trypsin + lysed with detergentd Intact cells + lysed with detergent

6.59 1.53 67.34 77.17

k 0.57 k 0.67 & 14.75 & 5.96

“Viability of cells 97.3%. ‘fi-1,4-GT activities were determined using ovalbumin as exogenous acceptor. Activities toward endogenous acceptors were subtracted; mean + SD of four determinations. ‘Cells were incubated with trypsin-EDTA for 10 min at 37°C; the reaction was terminated by the addition of an excess of ice-cold RPM1 1640 supplemented with 10% heat inactivated fetal calf serum and the cells, were then further washed with RPM1 1640. “Cells were solubilized with 1% Triton X-100 in 10 mM Tris-HCl. pH 7.2.

GlcNAc. The activity in the presence of cells (64.8 pmol of N-acetyllactosamine formed/5 x lo5 cells/h) was 5.6 x larger than that in cell-free medium (11.6 pmol/h). Ig as acceptor

substrates

of cell surface

fl-1,4-GT

The detection of radioactivity in Ig and their component chains following their incubation with live HT-29 cells in a medium containing Mn’+ and UDP-[3H]-Gal, indicated that these glycoproteins were galactosylated by the cell surface /3-1,4-GT. The amount of [3H]-Gal transferred to individual proteins decreased in the order: polymeric (p) IgA2 > monomeric (m) and pIgA > IgM

> IgG. Galactosylation of the isolated component chains was more effective than that of the entire Ig molecules. For example. J or c( chains (both from secretory (S)-IgA), were galactosylated to a higher extent than native S-IgA. Likewise, the p chain was a better acceptor than the native IgM. IgG and, surprisingly, carbohydrate-rich SC (Mizoguchi et al., 1982), were relatively poor acceptors (Table 3). Cell surjkxe p-1,4-GT In the presence Gal, B-1,4-GT

as a lectin

of MnCl, and absence of UDP-[3H]forms stable complexes with its

Fig. 1. Surface fluorescence (left) and phase contrast (right) of HT-29 cells. Live cells were incubated with rabbit antiserum to human p-1,4-GT and subsequently stained with TRITC-conjugated goat anti-rabbit IgG.

269

Cell-surface galactosyltransferase Table 3. Activities of HT-29 cell surface /l-1,4-GT toward various acceptors’ /?-1,CGT activity relative to ovalbumin

Acceptor J chain (from S-I& chain (from IgA2) Ovalbumin a chains (from S-Ig4) L chain fraction (containing J chain from S-I& p chain (from IgM Daub) Polymeric 1842 (Fel) Fc fragment (from IgM Dau) Monomeric IgAl (Pet) Monomeric IgAl (Kni) Dimeric IgAl (Ham) S-18A (from colostrum) Dimeric IgAl (Car) IgM (Lin) IgG (from patients with rheumatoid arthritis’) Secretory component (from S-&A) IgG (from healthy individuals) a2

2.12 1.61 1.00 0.51 0.29 0.19 0.18 0.13 0.11 0.08 0.06 0.05 0.04 0.04 0.025 0.020 0.020

“Each assay contained 5 x 10’ cells, 0.1 mg protein acceptor, and other components as described in Material and Methods. Data were corrected for the activity assayed in the absence of exogenous acceptors. “Dau, Pet, Kni, Fel, Car, and Ham signify specific patients. ‘IgG from pooled sera of four patients with rheumatoid arthritis had 34% less Gal than IgG from controls.

acceptor substrates. The interactions of /3-1,4-GT with Ig were examined by determining the ability of bovine milk /I - 1,4-GT, immobilized on Sepharose 4B, to bind Ig of various isotypes and molecular forms. Varying amounts of Ig, a2 chain and albumin (the last as a negative control) in a Mn2+ containing medium were passed through the column of bovine milk p-1,4-GTSepharose 4B. The bound protein was eluted with a medium containing EDTA. As shown in Table 4, fi- 1,4GT bound all three major isotypes of Ig; the portion of total protein bound varied from 53% for isolated a2 chain to 17% for native IgG. The binding of some of these Ig to /I-1,4-GT was proportional to their binding to a GlcNAc-specific wheat germ lectin. The binding of Ig to cell surface b-1,4-GT was visualized, using biotinylated a 2 and J chain as probes, by immunofluorescence staining. As shown in Fig. 2, live HT-29 and Hep G2 cells, which were incubated in a medium containing 10 mM MnCl, with biotinylated a2 or J chain, and subsequently with avidin-labeled AMCA displayed a prominent surface fluorescence. Controls included cells which were incubated with biotinylated IgA or a chain in a medium containing 10 mM EDTA instead of MnCl, and with avidin-AMCA alone. Specljicity of ligand binding to cell surface /I-1,4-GT To study the specificity of interactions of cell surface /I-1,4-GT with its substrates, the HT-29 cell line was used in conjunction with either the n&Al myeloma protein or the isolated a2 chain because these proteins

do not bind to SC expressed on surfaces of HT-29 cells (Crag0 et al., 1978). Furthermore, HT-29 cells do not display ASGPR (Milan Tomana, unpublished observation), which is responsible for binding of the above proteins by hepatocytes and Hep G2 cells (Tomana et

Table 4. Binding of Ig to bovine milk /?-l,CGT-Sepharose 4B and wheat-germ lectin (WGL)-Sepharose 6MB” Protein Heavy chain from IgA2 (Fel) Polymeric 1842 (Fe]) Monomeric IgAl (Pet) Polymeric IgAl (Car) Pentameric IgM (Dau) s-1&4 (colostrum) IgG (pooled human serum) Bovine serum albumin

/l-1,4-GT WGL Protein bound (%) 53 46 33 29 31 26 17 11

82 ND 75 ND ND ND 24 ND

“Varyingamounts of proteins (0.28-4.10 mg) in 25 mM sodium cacodylate, pH 7.2, containing 25 mM MnClr and 0.1 M NaCl were applied to a column (11 x 20 mm) of bovine milk /l-1,CGTSepharose 4B equilibrated with the same buffer. Proteins bound were eluted with 25 mM sodium cacodylate, pH 7.2, containing 25 mM EDTA and 0.1 M NaCI. An equal amount of the same protein in PBS was applied to a column packed with WGL-Sepharose 6MB (11 x 30 mm) equilibrated with PBS. Adsorbed protein was eluted with 1 mM GlcNAc in PBS. The amount of protein bound was estimated by measuring volume and the optical density at (280 nm) of the eflluent. The data shown correspond to the ascending part of the saturation curves.

270

M.

T~MANA

et

al.

Fig. 2. Surface fluorescence of HT-29 cells. Live cells were incubated J (right)

chains, washed and incubated performed at 0 C in an isotonic

with biotinylated a2 (left) and with AMCA. Staining and washing procedures *cre MES buffer, pH 7.2, containing 10 mM MnCl?.

al., 1988). The binding of ‘251-IgA by HT-29 cells was partially inhibitable with EDTA, GlcNAc, a-lactalbumin (in the presence of glucose), and uridine 3’,5’-dialdehyde. Quantitatively similar data were obtained when U937 cells were used (Fig. 3). The inhibition studies further revealed that the binding of tx2 chain to cell surface fi - 1,4-GT on HT-29 cells was dependent on the concn of MnCl,, which had to be in molar excess relative to GlcNAc. In the presence of 10 mM MnCl,, the maximum inhibition was observed at 1 mM concn of GlcNAc; further increase in the concn of GlcNAc resulted in a decrease of the inhibition (data not shown).

A

B

C D HT-29

E

The effect of the concentration of bivalent cations (Ca2”, which activates binding of suitable ligands by ASGPR, and Mn*+) on the binding of IgA to cells of HT-29 and Hep G2 cell lines or mouse hepatocytes was investigated to evaluate the relative involvement of fi- 1,4-GT and ASGPR. As shown in Fig. 4, the presence of Mn2+ promoted binding of IgA by both these cell lines and hepatocytes; the binding was mediated mainly by /3-l ,4-GT. In the presence of Cal+, the increase of the binding was observed only in Hep G2 cells and mouse hepatocytes, but not in HT-29 cells, indicating involvement of ASGPR, which is absent in HT-29 cells. In the presence of both of these ions, the binding of IgA by HT-29 cells was not significantly different from that in the presence of Mn’+ alone. Hep G2 and mouse

ABC u 937

Fig. 3. Inhibition of binding of mIgA1 to HT-29 and U937 cells. HT-29 and U937 cells fixed with glutaraldehyde were incubated with monomeric ‘2SI-IgAl (79 rig/assay)) in the presence of: 17 mM MnCl, (A); 17 mM EDTA (B); 17 mM MnCl, + 1 mM GlcNAc (C); 17 mM MnCl,+ 0.046mM tlglucose (D); 10 mM uridine lactalbumin + 10 mM 3’,5’dialdehyde (E).

D Hepatocytes

ABCD Hep-GP

Ii! ABCD

HT.29

Fig. 4. Effect of Mn*+ and Ca’ f on binding of IgA by cell surface b-1,4-GT. Cells fixed with glutaraldehyde were incubated with 13.4-17.6 nM monomeric IgAl in the presence I1 mM EDTA (B); MnCl: 11 mM 1lmM (A); MnC12 + 11 mM CaClz (C); 11 mM CaClz (D).

271

Cell-surface galactosyltransferase Table 5. fl-1,4-GT activity in suspensions of viable and glutaraldehvdefixed HT-29 cells

#.llS Viable Fixed

fl-1,4-GT activity toward” GlcNAc Ovalbumin (omoles nroductl2 x 10’ cclls/hrf

Q@-1,4-GTactivities were corrected for activities toward endogenous acceptors. hepatocytes express both, fi-1,4-GT and ASGPR; thus, in the presence of Mr?+ and Ca2+, both receptor should be activated. However, results of the binding experiments demonstrated that in Hep G2 the binding of IgA in the presence of both of these ions was approximately the same as if only one of them had been present; in hepatocytes the binding in the presence of both ions was lower than if only Ca2+ or Mn2’ had been present. These results indicated that in the presence of Mn2+, the binding of IgA by ASGPR was inhibited. Binding data on interactions of Ig with ceil surface /I-1,4-GT

Kinetic and equilibrium parameters of interactions of Ig with cell surface @-1,4-GT were studied with cells whose receptors were stabilized by mild fixation with glutaraldehyde (Williams, 1973). As shown in Table 5, fixed HT-29 cells exhibited an enzymatic activity comparable to unfixed cells. Fixed and unfixed cells exhibited also similar cell binding activity (data not shown). This treatment eliminated the problems of cell lysis and shedding of @-1,CGT into the medium, and facilitated separation of cells from the medium by centrifugation through oil (a mixture of dibutyl phthalate and dinonyl phthalate). Data concerning the apparent affinity constant (Kl) and association of rate constant (k + 1) were determined for interactions of m&Al and a2 chains with cell surface p-1,4-GT on glutar~dehyde-axed cells of

0.0 0.~ 1.0 $5 20 2s aa ad

4.0

4.3

6.0

6.5

HT-29 (fixed)

HT-29

u937 (fixed)

7.1 x 10’

ND

2.0 x 108

Kl [M-l]

29.2 + 2.2 31.7 It 4.0

3.9 * 0.4 4.2 f 0.3

Table 6. Binding data of interactions of a2 chains (Fel) with cell surface p-l>

:+ 1 [M-’ s-‘1 k - 1 Is-‘1 G/ZIi -

106 13.0 1.5 x 10s 2.1 x 1O-2 33

9.0ND x 105 ND ND

4.8 1.0 x lo5 105 5.0 x 10-4 1380

Kl, apparent affinity constant; n, number of a2 molecules

bound to one cell; k + 1, association rate constant; k - 1, dissociation rate constant; t,,2r half time of dissociation; ND, not determined. Concentrations of bound ligands used in calculation of binding constants were estimated from the difference of the binding in the presence of Mn2+ and in the presence of EDTA. The concns of ligands in the assay system were corrected according to their ability to bind to wheat germ lectin.

HT-29 and U937 cell lines. The number of /3-1,4-GT receptors per cell (n) was estimated from the Scatchard plot (Fig. 5) by determining the limit number of Ig molecules bound by fixed and native HT-29 and iixed U937 cells. Experimental values in the Scatchard plot were obtained from saturation curves prepared by incubating cells with varying concns of ligands for various time periods in media which contained either Mn2+ or EDTA. Concentrations of mIgA1 and a2 chain used in calculations of Kl and k + 1 were corrected for the fact that only a subpopulation of the molecules contains a terminal GlcNAc and is therefore capable of interacting with @-1,4-GT. The correction factors used (0.82 and 0.75 for 012chain and mlgA1, ~s~tively) were obtained by quantitating the interaction of Ig with GlcNAcspecific wheat germ lectin as shown in Table 4. Data in Tables 6 and 7 demonstrate that the apparent affinity constants of IgAl were higher than those of a2 chain (2.1-4.6 x 10’ and 7.1-20 x lo7 M-l, respectively) possibly due to bivalency of the m&Al protein. Likewise, association rate constants of interactions of mIgA1 with cell surface B-1,4-GT were higher than those of the ct2 chain (4.2-30.0 x 10’ and 1.0-15.0 x lO’M_’ s-l, respectively). The number of receptors on HT-29 cells (5.1-13.0 x 10’) was slightly higher than that on U937 cells (2.04.8 x 103, probably as a result of differences in cell size. The difference in the molecular size between mIgA and a2 chain may explain binding of larger numbers of a2 chain molecules both to HT-29 and U937 cells.

e.0

MxlrJ-@mouLf

Fig. 5. Scatchard plot analysis of the binding of ar2 chain to /?-1,4-GT on glutaraldehyde fixed HT-29 and U937 cells. Cells (2.5 x lO’/assay) were incubated with various concns of the ‘2sI-a2 chain in the presence of 17 mM MnCl, and 17mM EDTA. The cell-bound and free. a2 chains were separated by ~nt~fu~tion through a layer of dibutylphthalate and dinonylphtbalate (7:3). (b) Concentrations of bound a2 chain; (c) concentrations of free a2 chain. /I-l,CGT-bound a2 chain was estimated from the difference of binding in the presence of MnCl, and EDTA.

Table 7. Binding data of interactions of monomeric IaAl (Pet) with cell surface /l-l> HT-29 (fixed)

Kl [M-l] n k+ 1 w-’ s-l] k- 1 [s-l] t1/2[Sl

4.6 x 108 4.9 x 105 3.0 x 106 6.5 x 1O-3 106

U937 (fixed)

2.1 x 108 1.9 x 105 4.2 x lo5 2 x 10-3 345

Kl, n, k f 1, k - 1, tli2, See legend in Table 6.

272

M. TOMANA Edal.

The dissociation rate constants (k - 1) of interactions of IgAl with HT-29 and U937 cells, calculated from these data were 6.5 x low3 and 2.10-s s-‘, which correspond to a half time of dissociation (&) of 106 and 345 s, respectively. The dissociation rate constants of interactions of a2 chain with the above cell lines were more diverse (2.1 x lo-* and 5 x lo-” s-l), corresponding to t,,, of 33 and 1380 s respectively.

interacts with the potential acceptors of Gal to form stable complexes. Other glycosyltransferases, which have been detected in plasma membranes in the absence of corresponding sugar nucleotides may also behave as fectins (Shur, 1989); however, p-l,4-GT is by far the most active cell-surface glycosyltransferase (Shur, 19823). Acceptor substrates of /.I-1,4-GT could be either simple sugars such as GlcNAc and glucose (in the presence of ~-lac~lbumin), or glycoproteins and glycoThe fate of I~ga~~ hound to ceII surface B-1,4-GT lipids with terminal GlcNAc residues (Ram and Munjal, To determine whether ligands bound to cell surface 1985). Structural studies have shown that N-glycosidfi-1,4-GT are internalized and catabolically degraded, ically-linked oligosaccharide units of all major isotypes Hep G2, and HT-29 cells were incubated for 60 min at often lack terminal sialic acid and Gal, thus exposing 37°C in Mn’+-containing medium with ‘251-1abeled~12 terminal GlcNAc residues (Rademacher and Dwek, 1984; Ohbayashi et al., 1989; Wold et al., 1990). The chains and mIgA1, then washed and lysed in a buffer deficiency in glycosylation may be associated with their containing 2% SDS. The lysate was subjected to analysis by SDS--PAGE The distribution of the radioactive high rate of synthesis when compared with other glycomarker in the gel was similar to that of control “‘I-m2 proteins (Nishiura et al., 1990). Braun et al. (1976) and ‘251-mIgAl, which were not incubated with cells, reported that during exponential increase of antibody indicating that the p-l,4-CT-bound ligands were not production, one cell is capable of secreting 2.4 x lo4 Ig molecules/set. In IgG isotype the deficiency of Gal is degraded. To further determine whether the /I-1,4-GTbound ligands were internalized, HT-29 and Hep G2 increased in certain chronic inflammatory diseases and in cells were incubated with ‘251-~2 and ‘2SI-mIgAl as aged individuals (Parekh et al., 1988; Tomana et al., 1988b). Data presented in this paper (Table 3) show that described above. Then the cells were washed and normal IgG as well as Gal-deficient IgG were relatively incubated for 10 min with trypsin-EDTA. Trypsinization, which was terminated by the addition of FCS, poor acceptors when compared with Ig of other isotypes. and further washing with isotonic buffer, resulted in This may be associated with poor accessibility of the removal of ~75% of cell-associated radioactivity. The terminal GlcNAc, previously suggested by crystallotrypsinized cells, which were still >90% viable, were graphic studies (Sutton and Phillips, 1982). It is also lysed with SDS-containing medium and analyzed by possible that galactosylation of IgG is mediated by SDS-PAGE. The distribution of the radioactivity in p-1,4-GT with a narrow specificity, such as /I-1,4-GT found in IgG-producing I3 cells (Furukawa et al., 1990), gels, revealed that both proteins were proteolyti~ally degraded. The absence of an appreciable amount of which may be absent in other cell types. The most intact ligands indicated that they were present on the cell effective acceptor of Gal of the three major isotypes tested was IgA, and IgA2 in particular. This is consistent surface rather than in the cytoplasm, where they would with the relatively large number of N-glycosidicallybe protected from proteolytic degradation by trypsin. linked oligosaccharides in IgA2 (Torano et al., 1977). These results do not support the concept that surface Carbohydrate structural analysis revealed that large ~-1,4-GT-bound ligands are internalized. number of terminal GlcNAc residues in the IgA2 (Fel) is not galactosylated (Wold et al., 1990). Component DISCUSSION chains of Ig (tl, p, and J chain) were better acceptors of Gal than native Ig (Table 3). This observation suggests Data presented in this report indicate that fi-1,4-GT that the terminal GlcNAc residues in isolated comis expressed in an enzymatically active form on plasma membranes of mo~hologically and functionally differ- ponent chains are better accessible for interaction with the enzyme than those in assembled Ig molecules. It is ent cells including mouse hepatocytes, human hepatoma likely that interactions of B-1,4-GT with Ig molecules cell line Hep G2, human colonic epithelial adenocarcinema cell line HT-29, monocyte-like cell line U937, as are hindered by lack of space on the cell surface. Ig and their component chains which were good acceptors well as human B and T cells. The presence of cell surface /I-1,4-GT was indicated by the observations that: (a) in of Gal when incubated in the presence of live cells the presence of live cells t3H]-Gal was transferred from (Table 3), were also efficiently bound to /I-1,4-GT immobilized on Sepharose 4B (Table 4). However, while UDP-[3Hl-Gal to exogenous and endogenous acceptors; the efficiency of galactosylation in the presence of live (b) the /I-1,4-GT activity could be removed by trypsinizcells rapidly decreased with increasing molar mass of ation and (c) a prominent surface immunofluorescence was observed when cells were stained with the use acceptors, the binding to p-1,4-GT-Sepharose was only of anti-p- 1,4-GT antibodies. These findings indicate that moderately affected by the molar mass of acceptors. This in the presence of UDP-Gal, cell surface /I-1,4-GT is may be due to a low density of fi-l,4-GT molecules per surface area of the Sepharose beads when compared to able to perform the same functions as #i-l ,4-GT located in the Golgi apparatus (Paulson and Colley, 1989). that on the cell surface. Interactions of fl-1,4-GT with Ig required the presence of Mn2” (a cofactor of all glycosylHowever, UDP-Gal is not normally present in extratransferases) (Weiser, 1973) and could be inhibited by cellular fluids and under these conditions fi-1,4-GT

273

Cell-surface galactosyltransferase chelating agents such as EDTA. The binding of Ig to cell surface /?-1,4-GT could also be competitively inhibited with small molar mass substrates such as GlcNAc, a-lactalbumin with glucose, or uridine 3’,5’-dialdehyde. The high effectiveness of inhibition with EDTA relative to other inhibitors (Fig. 3), could be caused by the presence on the cell surface of another glycosyltransferase or lectin which is activated with Mn*+. To study the kinetics of interactions of Ig with cell surface /l-1,4-GT, the stability of /3-1,4-GT-Ig complexes and the fate of /I-1,4-GT-bound ligands, it was necessary to eliminate the effect of other Ig-binding receptors. Earlier studies have shown that different cell types express diverse receptors for Ig (Mestecky and McGhee, 1987; Mestecky ec al., 1988). With respect to human IgA, the two molecular forms, mIgA and pIgA, also differ in their ability to interact with receptors on epithelial cells, monocytes and T cells (Brandtzaeg, 1985; Mestecky and McGhee, 1987; McGhee et al., 1989). By using populations of cells that express /l-l ,4-GT but lack SC (U937, Hep G2) or ASGPR (HT-29), and by manipulating the conditions which optimize or inhibit the binding through a given receptor, we were able to evaluate interactions of specific ligands with /I-1,4-GT. For example, using m&A, a2, or J chain eliminated the binding by SC whereas the binding by ASGPR was inhibited in the presence of high concentrations of Mn*+ (Fig. 4). Values of apparent affinity constants of interaction of cell surface /3-1,4-GT with IgA proteins which ranged from 7 x lo7 to 5 x 10’ M-l, indicate that the complex /I-1,4-GT-IgA is relatively stable. The dissociation constants of IgAl bound to HT-29 and U937 cells, 6.5 x lop3 s-’ and 2 x 10m3s-‘, respectively, are similar to those reported for interactions of agalactoorosomucoid with GlcNAc-specific receptors from chicken liver (1.3 x lop3 s-l) (Kawasaki and Ashwell, 1977) and asialoorosomucoid with human hepatic lectin (1.7 x 10m3s-‘) (Baenziger and Maynard, 1980). Likewise, the number of Ig molecules bound at extrapolated limited concentrations to surface /l-1,4-GT of a single cell of HT-29 and U937 cell lines (1.9 x lo’--1.3 x 106) is comparable or higher than that of ASGPR on Hep G2 cells (2.25 x IO’) (Schwarz et al., 1982). The existence of receptor(s) specific for various molecular forms of IgA, in addition to SC and ASGPR was predicted in earlier in vitro studies with human and rodent hepatocytes (Tolleshaug et al., 1981; Brandtzaeg, 1985; Mestecky and McGhee, 1987). Although their biochemical identity and binding capacity have not been determined, our current studies indicate that /?-1,4-GT may be one of them. The in vivo significance of our finding is at present unknown. Although the concn of Mn*+ in human body fluids is relatively low, other bivalent cations such a Cd*+, Mg2+, Ca*+, Fe2+, or Co*+ (Babad and Hassid, 1966; Podolsky et al., 1977; Cheng and Bona, 1982) may also, with lower efficiency, activate /3-1>. In vitro the optimum concn of Mn*+ , which is dependent on the concn of the acceptor, ranged from 2 to 43 mM (Ram and Munjal, 1985). In vivo cell-cell interactions

mediated by cell surface fi-l+GT, which occur during fertilization and embryonal development (Shur, 1989), the presence of circulating fi-l>-Ig complexes in external secretions and serum (Tomana et al., 1991) or galactosylation processed within the cell (Ram and Munjal, 1985) indicate that /I-1,4-GT is active as an enzyme or as a lectin even at a low concn of Mn*+, possibly due to the contribution of other bivalent cations. Cell surface glycosyltransferases have been considered as important participants in contact-mediated cellular interactions. The role of glycosyltransferases on cell surfaces was compared to that of lectins, because of their ability to interact with surface glycosides (Ram and Munjal, 1985). In this study, we report interactions of the most active cell surface glycosyltransferase, B - 1,4-GT (Shur, 1975) with Ig. b-1 > on hepatocytes, monocytes, and other cells which are in contact with blood, is mainly binding IgG, because of its high concentration in plasma. In contrast, on epithelial cells which are in contact with external secretions, /3-1,4-GT most likely binds preferentially IgA. Certain properties of cell surface /I - 1>, including the high number per cell, ability to bind Ig and the stability of the /I-l>-Ig complexes are similar to receptors, such as ASGPR (Baenziger and Maynard, 1980) or avian hepatic binding protein specific for GlcNAc (Kawasaki and Ashwell, 1977) or Fc receptors. However, the physiological role of surface fi-1,4-GT is unknown. Results of this study indicate that /?-1,4-GT is not involved in the internalization and catabolism of bound ligands. A possibility cannot be ruled out that @-1,4-GT may represent an Fox receptor which has not been characterized on all &A-binding cell types. In addition to p-1,4-GT, SC and ASGPR, other, as yet unidentified, IgA receptor(s) may be present on cells used in this study because binding of various molecular forms of IgA and c1chains to different cell types occurred even in the presence of specific inhibitors. Acknowledgements-This work was supported by Grants DK 28537 and AI 10854 from the National Institutes of Health.

The authors wish to express their appreciation to Dr Fiona Hunter for valuable criticism, Mrs Rhubell Brown for her technical assistance and to Mrs Maria Bethune and Mrs Linda Tomana for editorial help in preparing the manuscript. REFJZRENCES

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