THE JOVRNAL UF CHE41fSTRY Vol. 269, No. 15, Issue ofApril ... · G-10 (PD 10 column, Pharmacia) under red light. The purity of the '251-labeled product was checked by thin layer chromatography

THE JOVRNAL UF BIOLOGICAI. CHE41fSTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 15, Issue ofApril 15, pp. 11327-11336, 1994 Printed in U.SA.

~hotoide~t i f i~at io~ of Mannosyltransferases of Dolichol Cycle in the Mammary Gland PURIFICATION AND CHARACTERIZATION OF GDP-Man:Man@l- 4GlcNAcP1 -j 4GlcNAc-P-P-DOLICHOL MANNOSYLTRANSFERASE"

(Received for publication, December 21, 1993, and in revised form, February 3, 1994)

Ashok ~udgapalli , Samir K. Roy, Eric H. Holmes% and Inder K. ViiayS From the ~ e p a r t ~ e n ~ of Animal Sciences, University of M a ~ l a n d ~ College Park, M u ~ ~ a n d 200742 and the $Pucific Nor~hwes~ Research Fo~ndution, Seattle, Was~in~ton 981224327

GlcJMan,GlcNAc,-P-P-Dol serves as the major precursor for the biosynthesis of asparagine-linked glycoproteins in eukaryotes. The first 5 of the 9 mannosyl residues during the assembly of the oligosaccharide moiety within the dolichol cycle in the endoplasmic reticulum are incorporated directly by the action of GDP-Man-requiring mannosyltransferases while the remaining last 4 mannosyl residues are transferred by Man-P-Dol-requiring enzymes. In an earlier study (Shailubhai, K., Illeperuma, C., Tayal, M., and Vijay, I. K. (1990) J. SioZ. Chem. 265,1410~14108), we identified the enzyme UDP- G1c:Dol-P glucosyltransferase by ~hotolabel~ng rat mammary microsomes with 5.N~"[P-a2PJUDP-Glc. Apply- ing a similar strategy, GDP-he~anolamine-~~&azidosalicylic acid, an analog of GDP-Man, was found to photolabel two polypeptides of 31 and 69 kDa among the microsomal proteins of the rat mammary gland. A differ- ential ammonium sulfate saturation ~60-800/0~ of the detergent-solubilized microsomal proteins enriched the 69-kDa polypeptide. Pho~label~ng of this polypeptide was specifically inhibited by anin ne-containing nucleotides and nucleotide-sugars and was associated with a GDP-Man-requiring mannosyltransferase. The mannosyltransferase was purified nearly 16,000-fold and shown to contain the 69-kDa polypeptide.

The purified enzyme catalyzes the transfer of ['*C]Man from GDP-["C]Man to ManPl -+ 4GlcNAcPl + 4GlcNAc-P-P-Dol in al,3-linkage to give [14C]Manal -+

3ManPl 4GlcNAcPl -+ 4GlcNAc-P-P-Dol as the product. Antibodies raised against the 69-kDa polypeptide removed the enzymatic activity from the detergent ex- tract of the rat mammary microsomes and reacted specifically with a polypeptide band of the same size on immunoblots. The purified enzyme showed a pH optima of 7.4-7.8, Ir, - 4 pm for GDP-Man, -2-fold activation by phosphatidylchol~e, and a strong inhibition by sulfhydryl-selective reagents, N-ethylm~eimide and p-chloromercuribenzoate. The availability of the highly purified enzyme and a monospecific antibody should allow its molecular cloning for investigating the regulation of the machinery for protein N-glycosylation upon hormonally

* This work was supported by National Institutes of Health Grant DK19682, a Distinguished Faculty Research Fellowship (to 1. K. V.) from the Graduate School, and Grant RFP-ANSC 92-10 from the Mary-

tribution No. 8761, article no. A-6550 of the Maryland Agricultural land Agricultural Experimental Station. This paper is Scientific Con-

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modulated growth and differentiation of the mammary gland during its ontogeny.

Asparagine-linked glycoproteins represent the largest class of glycoproteins and possess diverse and versatile oligosaccharide moieties whose structures are consistently and faithfully reproduced by the eukaryotic cell even though no template is used to construct them. The biosynthesis of the precursor carbohydrate unit of these proteins is initiated by a stepwise assembly of Glc~Ma~GlcNAc~-P-P-Dol' in the dolichol cycle, its transfer en bloc to the nascent polypeptide in the RER, followed by excision of the glucosyl residues by processing-specific enzymes, glucosidase I and 11, also resident in the ER. Additional post-translational modifications of the carbohydrate moiety in the RER, GoIgi, and the trans-Got@ network for the completion of final products as high mannose, complex or hybrid glycoproteins en route to their Enal destinations in the secretory pathway differ for individual glycoproteins (1).

A differentia^ expression of an ensemble of more than 100 glycosyltransferases and glycosidases is required for the elabo- ration of the extensive repertoire of glycoconjugate structures in the mammalian cell. Within the ER, a concert of up to 17 glycosyltransferases and 2 glycosidases, glucosidase I and 11, is minimally essential for the biosynthesis of all-N-linked glycoproteins. For the assembly of Glc,Man,GlcNAc,, the first 5 mannosyl residues are sequentially transferred directly from GDP-Man to GlcNAc,-P-P-Dol to give rise to Man,GlcNAc,-P- P-Dol. Subsequently, 4 mannosyl residues are added, also sequentially, via the intermediary formation of Man-P-Dol from dolichol-P and GDP-Man, to build the intermediate Man,GlcNAc,-P-P-Dol. Finally, 3 glucose residues are transferred from Glc-P-Dol to yield the precursor Glc~Man~G1cNAc~-

The ~ ~ - l o c a l i z e d glycosyltransferases of the dolichol cycle are in low abundance and are highly labile enzymes that have proven far more refractory to purification than the glycosyltransferases in the Golgi complex and the trans-Golgi network. Only limited and partial purifications have been achieved for several of these enzymes (3-18). Nevertheless, we reported the first homogeneous purification of an enzyme of the dolichol cycle, i.e. UDP-G1cNAc:Dol-P-GlcNAc-1-P-transferase, the enzyme at the commitment step for protein N-glycosylation (19).

The abbreviations used are: Dol, doiichol; Glc, glucose; Man, mannose; GlcNAc, ~~-acety~glucosamine; Fuc, fucose (fucose has an L-con- figuration whereas all other sugars have the ~ - c o n ~ ~ r a t i o n ) ; RER, rough endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; endo D, endo-~-N-acetslg~ucosaminidase D; endo L, endo-8-N- acetylglucosaminidase L; ASA, azidosalicylic acid; AMP-CPP, adenosine 5'-(n,/3-methyleneftriphosphate.

P-P-Dol(1, 2) .

11327

11328 Mannosyltransferase of the Dolichol Cycle

The enzyme GDP-Man:Dol-P mannosyltransferase from Sac- charomyces cerevisiae was subsequently purified to homogeneity (20). Recently, Glc,Man,GlcNAc,-P-P-Do1:acceptor peptide oligosaccharyltransferase was shown to comprise a complex of ribophorin I and I1 and a 48-kDa polypeptide in the ER of animal tissues (21, 22), and a similar complex of Wbplp and Swplp protein in the yeast (23, 24).

Photoaffinity labeling of microsomal proteins with analogs of sugar-nucleotide substrates offers a novel approach to tag the catalytic polypeptides of glycosyltransferases prior to purification. Such a labeling of the partially purified preparations of the solubilized microsomal proteins with 5-azid0-[p-~~P]UDP- Glc identified a 35-kDa polypeptide as representing UDP-Glc: Dol-P glucosyltransferase in the rat mammary gland (16), liver (25), and S. cerevisiae (26). The photoactive reagent GDP-hexanolamine-4-azidosalicylic acid, an analog of GDP-Man and GDP-Fuc identified GDP-fucose:nLcOs,Cer al,3-fucosyltransferase from the human small cell lung carcinoma NCI-H69 cells (27). Using the same reagent, we now report that two polypeptides of 69 and 37 kDa are specifically labeled in the microsomes of the rat mammary gland. The 69-kDa polypeptide represents the catalytic component of the enzyme GDP-Man: Manpl - 4GlcNAcpl - 4GlcNAc-P-P-Dol a1,3-mannosyltransferase. After solubilization of the membranes with 0.5% Nonidet P-40, this enzyme has been purified to near- homogeneity; a number of its properties have also been characterized.

EXPERIMENTAL PROCEDURES Material~-GDP-['~CImannose (284 mCi/mmol) was purchased from

DuPont NEN. Carrier-free Na'"1 was obtained from Amersham. AMP- CPP, dithiothreitol, phospholipids, concanavalin A-Sepharose, and com- mon reagents were the products of Sigma; 2,3-dimercapto-l-propanol was purchased from Aldrich; Sephacryl S-300 was from Pharmacia LKB Biotechnology Inc. Electrophoresis reagents and protein A-agarose were purchased from Bio-Rad Laboratories; IODO-BEADS were obtained from Pierce Chemical Co. The lactating bovine mammary tissue was obtained from a local slaughterhouse, frozen in Dry Ice, and stored at -80 "C until used. Mammary glands of lactating rats were supplied by Hilltop Laboratory Animals, Inc. (Scottsdale, PA). a-Mannosidase and endo D were the products of Genzyme; alkaline phosphatase substrate was purchased from Vector Laboratories. Alkaline phosphatase-conju- gated, anti-rabbit IgG antibodies were purchased from Promega Biotec; DEAE-Trisacryl was from IBF Biotechnics. GDP-hexanolamine-4- azidosalicylic acid (ASA) was prepared as before (27). GDP-mannuronic acid was prepared according to the method of Roychoudhury et al. (28). Mlnity matrix GDP-hexanolamine-Sepharose was a kind gift from Dr. Ole Hindsgaul, University of Alberta, Canada; Dr. Robert Trimble, NY State Dept. of Health, Albany, N Y , generously gave the enzyme endo L. Radioactive [Man-3Hloligosaccharide standards ManPl + 4GlcNAcPl --f 4GlcNAc, Manu1 + 3ManPl+ 4GlcNAcpl- 4GlcNAc, Mana l - 3(Manal- 6)Manpl- 4GlcNAcpl- 4GlcNAc, Manal + 2Manal- 3(Manal --f 6)Manpl - 4GlcNAcPl - 4GlcNAc, and Manal +

2Manal- 2Manal- B(Mana1- 6)Manpl- 4GlcNAcPl- 4GlcNAc were the products of this laboratory (29, 30).

Preparation of Solubilized Microsomal Proteins-The buffers used for tissue homogenization and the washing of microsomes contained antiproteases, o-phenanthroline (1 mM), phenylmethylsulfonyl fluoride (1 mM), and E-aminocaproic acid (1 m ~ ) . The washed microsomal preparation of the lactating rat mammary gland was prepared as before (31). The membrane pellet was resuspended in 50 mM HEPES, pH 7.2, containing 20% glycerol, 0.1% 2-mercaptoethanol, and 1 mM benzamidine (buffer B) to a protein concentration of 15-20 mg/ml. The membrane suspension was solubilized with the nonionic detergent, Nonidet P-40, a t a proteiddetergent ratio of 1 .51 with the final detergent concentration at 0.5%. After stirring for 15 min at 4 "C, the mixture was centrifuged a t 147,000 x g for 60 min in Ti-50 rotor of Beckman ultracentri- fuge. The supernatant was used as the source of solubilized enzyme.

Preparation of Stripped Microsomal Membranes-The microsomal suspension in buffer B was diluted to 8 mg/ml and treated with 0.5% (wh) saponin containing 150 m~ KC1 for 30 min a t 20 "C. It was then centrifuged at 48,000 x g for 1 h. The pellet representing the membranes devoid of their luminal contents and peripheral proteins (32,331

was resuspended in buffer B. Radioiodination of GDP-henunolamine-4-azidosalic~lic acid-It was

conducted as reported earlier (27). After the reaction, free iodine was removed by desalting the reaction mixture on a column of Sephadex G-10 (PD 10 column, Pharmacia) under red light. The purity of the '251-labeled product was checked by thin layer chromatography on silica gel in 95% ethanoVl M ammonium acetate, 7:3, followed by autoradiography. The product was used for photoaffhity labeling of the microsomal proteins.

Photoaffinity Labeling of Microsomal Proteins-Photoaffinity labeling was conducted in a reaction mixture consisting of 40 mM HEPES, pH 7.2, 20% glycerol, 2 mM EDTA, 5 mM CDP-choline, 60 nmol of GDP- hexanolamine-4-azido[12511~alicylic acid, and 100 pg of protein in a total volume of 0.2 ml. The mixture was incubated at room temperature for 30 min in the dark followed by photolysis a t 254 nm with a hand-held W-lamp for 90 s from a distance of 1.5 cm. The reaction was stopped by the addition of 0.2 ml of ice-cold 7% trichloroacetic acid. For competition experiments, 600 nmol of different nucleotides, sugar nucleotides and other potential competitors, were added to the reaction mixture, allowed to equilibrate for 5 min with the microsomal proteins a t room temperature before the addition of GDP-hexanolamine-4- azido['2sIlsalicylic acid and subsequent photolysis.

The trichloroacetic acid-treated mixtures were left on ice for 2 h and then centrifuged a t 10,000 x g for 15 min. The pellet was washed with 1 ml of -20 "C acetone, dissolved in the Laemmli sample buffer, and subjected to 10% SDS-PAGE. The gels were dried and exposed to x-ray film (X-OMAT, Kodak) a t -70 "C for 12 h.

Preparation of Lipid-linked Oligosaccharide Acceptors-These were prepared from 1.7 kg of bovine mammary tissue as described (31, 34). The glycolipids obtained were resuspended by sonication in 23 ml of 0.1% Nonidet P-40. The phosphate concentration of the micellar suspension was 88 pg/ml. It was aliquoted in 0.5-ml quantities in Eppen- dorf' tubes and stored at -80 "C. Just before use, the suspension was briefly sonicated.

Assay of Mannosyltransferases-The mannosyltransferase activities

HEPES, pH 7.6, 10 mM dithiothreitol, 5 mM MgCl,, 0.3 mMAMP-CPP, 5 were assayed using a standard reaction mixture that contained 50 mM

mM 2,3-dimercapto-l-propanol, 5 mM AMP, 0.05 pCi of GDP-['4ClMan, lipid-linked oligosaccharide acceptors (2.3 pmol of phosphate), and the enzyme fractions (up to 80 pg of protein) obtained a t different stages of purification in a final volume of 0.1 ml. After incubation at 37 "C for 30 min, the reactions were quenched with 2 ml of CHCl,/CH,OH, 2:1, and processed by the multiple extraction procedure to get products soluble in CHCldCH,OH/H,O, 10:10:3 (34). Careful attention was paid to ensure that no radioactive products other than [14ClMan-P-Dol were in the chlorofodmethanol, 2:l. This happens if the ratio of the two sol- vents changes from 2.00:l.OO and increases in its polarity such that lipid-linked oligosaccharides larger than GlcNAc,-P-P-Dol would parti- tion in both chlorofodmethanol. 2:1, and chloroform/methanol/water, 10:10:3, during the multiple extractions.

Preparation of [Man-'4C101igosaccharide Product-Scaled up incubations of the standard assay with the purified enzyme were carried out and processed to get quantities of the radioactive glycolipid soluble in chlorofordmethanollwater, 10:10:3. I t was dried down and hydrolyzed with 0.01 N HC1 at 90 "C for 60 min. Subsequent operations to get the desalted [Man-'4Cloligosaccharide were carried out as before (28).

Product Analysis-The [Man-'4Cloligosaccharide was subjected to descending paper chromatography in butanoVpyridine/H,O, 4:3:4 (system A) along with radioactive oligosaccharide standards for size deter- mination. It was digested with endoglycosidases D and L and a-mannosidase, and the products were analyzed by paper chromatography in either solvent system A or ethyl acetate/pyridine/acetic acidwater, 5:5:1:3 (system B).

Gel Electrophoresis-SDS-PAGE was conducted under reducing conditions as before (16). Gels were dried and exposed to Kodak X-OMAT film at -70 "C for 16 h. The Western blot analysis was conducted as before (19).

For native gel electrophoresis, several wells of the 7.5% gel slab prepared with 10 mM Tridglycine buffer, pH 8.3, containing 10% glycerol, were loaded with the solubilized microsomal proteins mixed with native gel buffer, 50 mM Tris-HCl, pH 6.8, containing 5% glycerol and 0.002% bromphenol blue. An adjoining well was loaded with an aliquot of the solubilized microsomal proteins that had been photolabeled with GDP-hexanolamine-4-azido[12sIlsalicylic acid, and mixed with the native gel buffer. The electrophoresis was conducted at 100 V at 4 "C. After the dye front reached the bottom of the gel, the run was stopped, and lane 1 was sectioned into 0.5-cm segments along the direction of migration. Each section was homogenized in 1.0 ml of 50 mM HEPES buffer,

Mannosyltransferase of the Dolichol Cycle 11329

pH 7.4, containing 20% glycerol, 1 my EDTA, and 0.5% Nonidet P-40 (vh) in a 1.5-ml glass homogenizer. The homogenate-containing tubes were shaken end-over-end on a rotator overnight a t 4 "C. The tubes were centrifuged a t 10.000 x g for 15 min. The supernatant was collected for measurement of mannosyltransferase activity.

Preparation of Antibodies-Antibodies against the rat mammary mannosyltransferases were raised in a New Zealand White female rabbit as follows. Active fractions of the enzyme eluting from GDP-hexanolamine-Sepharose were pooled and dialyzed against 5 mw Tris-HCI, pH 7.0, containing 1 m%1 phenylmethylsulfonyl fluoride. An aliquot was photolabeled with GDP-hexanolamine-l"'IIASA while the remainder was lyophilized. Both the photolabeled and the lyophilized enzyme were subjected to 10% SDS-PAGE in multiple runs. The resolved protein was transblotted onto nitrocellulose paper and autoradiographed. The seg- ment of the strip in the sample lane adjoining the photolabeled band was cut out and soaked in 50 mxl Tris-HCI, pH 9.0, 2% SDS, and 1% Triton X-I00 for 2 h a t room temperature on a shaking platform. More than 85% of the protein could be eluted from the nitrocellulose paper by this method. The eluted protein was dialyzed, with several changes. against 20 my Tris-HCI, pH 7.4, 10% glycerol for 40 h. The purity of the protein was confirmed on SDS-PAGE and by two-dimensional electrophoresis (35). The dialyzed protein was concentrated by lyophilization. About 100 pg of the protein (0.5 ml) was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously in multiple sites on the back of a rabbit. Booster doses of protein (50 pg) were given a t 2-3-week intervals for a total of three times. Blood was collected on the 10th day after the last booster. The antiserum obtained following the clotting of blood, after appropriate dilution, was used to probe the Western blots. For immunoinhibition studies, the antiserum was partially purified to obtain the I& fraction (19).

Immunoinhihition-The partially purified I& fraction of the antiserum was serially diluted with phosphate-buffered saline and incubated with 75 pg of rat mammary microsomal protein, solubilized with 50 mM HEPES, pH 7.4, 1 my EDTA, 5 m.cr 2-mercaptoethanol, 5 mu MgCI,, 20% glycerol, 1 mk! benzamidine, 0.59 Nonidet P-40 (v/v), or the dialyzed 6 0 4 0 % ammonium sulfate saturation fraction of the same for 4 h a t 4 "C. Protein A-agarose (50 pl of wet gel), washed and equilibrated with the above buffer, was added and mixed end-over-end on a rotator for 4 h a t 4 "C. The mixture was centrifuged and the enzyme activity was assayed in the supernatant as given above. Control experiments with preimmune serum were processed identically and carried out alongside.

Analytical Methods-Protein was determined by the BCA method (Pierce) with bovine serum albumin as the standard. Organic phosphate was measured by the method of Ames and Dubin (36).

RESULTS

Mannosyltransferase Activities in Differentially Solubilized Microsomes-We had earlier shown that 6 mannosyltransferases for the synthesis of intermediates up to Man,GlcNAc,- P-P-Dol and Man-P-Dol could be solubilized from the bovine mammary gland with several nonionic detergents; among these, Nonidet P-40 at a final concentration of 0.5% (v/v) gave optimum solubilization of the above enzymes (36). Prior to un- dertaking a photoaffinity labeling and making an attempt to purify one or more enzymes from the rat mammary gland, we confirmed the above results. For best results in terms of solubilization and stability, a proteiddetergent ratio of 1.5:l was chosen to get consistent data.

Photoaffinity Labeling of Intact, Stripped, and Solubilized Microsomes with GDP-hexanolamine-['"'I]ASA-When a preparation of microsomal proteins was incubated with photoactive reagent for 30 min at room temperature followed by a 90-s exposure to UV light, two polypeptides of 69 and 37 kDa, as shown by SDS-PAGE autoradiography of the products, were specifically labeled (Fig. 1). The relative intensity of the label in the 69-kDa polypeptide was much higher than that in the 37- kDa polypeptide (Fig. 1, lane 2) . The same pattern of labeling was observed when the microsomal proteins were solubilized with Nonidet P-40 or if their luminal and peripheral proteins were removed by treatment with saponin and KC1 (Fig. 1, lanes 3 and 4 ) . These results show that potentially two GDP-Man- requiring mannosyltransferases or binding proteins in the mi-

teins with GDP-hexanolamine-[ i251]ASA. One hundred micrograms FIG. 1. Photoaffinity labeling of rat mammary microsomal pro-

of protein in the different fractions was labeled with 60 nmol of the radioactive probe and subsequently processed as described under "Ex- perimental Procedures." The labeled proteins were subjected to 10% SDS-PAGE followed by the drying of the gel and autoradiography to detect the labeled polypeptides. Lane I , molecular size markers; lane 2, intact microsomes; lane 3, solubilized microsomes; lane 4, intact microsomes after treatment with 0.5% ( d v ) saponin and 150 mM KC1 to obtain membranes free of luminal and peripheral proteins.

crosomes recognize the sugar-nucleotide analog. Further, either the 69-kDa polypeptide has a greater affinity for the photolabel or is much more abundant than the 37-kDa polypeptide. Both proteins are also readily solubilized by the nonionic detergent, Nonidet P-40, maintaining the relative intensity of their photolabeling characteristics. The observation that both polypeptides are labeled in the same proportion in stripped microsomes as the intact membranes indicates that these are integral rather than peripheral membrane proteins.

UV and 7ime Dependency of Photoaffinity Labeling-The solubilized microsomal proteins were incubated with GDP-hexan~lamine-['~~II]ASA for different time periods followed by photolysis for 90 s. The results in Fig. 2 show that there is a gradual increase in the labeling of both 69- and 37-kDa polypeptides for time periods up to 30 min. There is no incorporation of the photolabel into the two polypeptides in the absence of exposure to UV light. It is possible that a more prolonged incubation of the solubilized microsomal proteins with the probe might have labeled additional proteins that might bind it with lower affinity. Similarly, an exposure to W light of more than 90 s might have revealed the labeling of more polypeptides. However, this would also risk a rather nonspecific labeling of proteins by the highly reactive nitrene released upon photolysis. Based on these observations, a 30-min incubation of microsomal proteins with GDP-hexanolamine- [IzeI IASA at room temperature followed by a 30-s exposure to a hand-held lamp emitting UV light of 254 nm from a distance of 1.5 cm was chosen as the standard condition for further photolabeling studies.

Separa.tion of Photolabeled Solubilized Proteins upon Native PAGE-The photolabeled solubilized microsomal proteins were separated by 7.5% native gel electrophoresis a t pH 8.3. The autoradiogram of the gel showed two labeled spots with rela-

11330 Mannosyltransferase

FIG. 2. UV and time dependency of photoaffinity labeling. The solubilized microsomal proteins were incubated with the radioactive probe for different periods of time followed by a 90-s exposure to W light (254 nm) with a hand-held unit from a distance of 1.5 cm. Lanr 1 , molecular size markers; reaction tubes for lanes 3-7 and 2 were incubated with the probe for 0, 10, 15.20.25, and 30 min, respectively, prior to W exposure. The tube in lane 8 was incubated for 30 min but not subsequently exposed to UV light. The samples were subjected to 10% SDS-PAGE and autoradiographed as before.

tive intensity similar to that observed earlier by SDS-PAGE (Fig. 3, lower panel ). Several parallel lanes of the native gel electrophoresis run alongside were sliced into 0.5-cm segments. The proteins in the pooled individual segments were eluted and assayed for mannosyltransferase activities in incubations containing GDP-["C ]Man and the mixture of Dol-P-P-linked saccharides extracted and partially purified from bovine mammary microsomes. The products of the reactions were obtained by multiple solvent extraction and quantitated by scintillation counting. As shown in Fig. 3, upper panel, five peaks of radioactivity can be clearly discerned. The dolichol cycle for the assembly of the GIc,Man,GlcNAc,-P-P-Dol contains five enzymatic steps in which GDP-Man serves as the immediate donor of the mannosyl moiety to dolichol-P-P-linked oligosaccharide acceptors (1). Further, the solubilized membranes synthesize only a small proportion of Man-P-Dol unless supplemented with Dol-P (35). It is, therefore, noteworthy that five peaks of radioactivity in Fig. 3, upper panel, may represent the GDP- Man-requiring mannosyltransferase activities of the dolichol cycle in the mammary gland. Unfortunately, the enzymatic activities are extremely labile and are not readily amenable for purification by the conventional methods of enzymology. These data lend strong support to the tentative inference that the 69- and 37-kDa polypeptides that are photolabeled by GDP-hexanolamine-[""IIASA represent two of the mannosyltransferases rather than GDP- or GDP-Man-binding proteins. By the same token, it would appear that the probe is unable to get into or orient its photolabile center close enough to the active site of the other mannosyltransferases such that the nitrene generated after photolysis could covalently insert into these enzymes.

Competition. Experimen.ts-Preliminary experiments indicated that the solubilized microsomal proteins could be differ-

of the Dolichol Cycle

entially precipitated with retention of photoaffinity labeling characteristic of GDP-hexanolamine-['z51 IASA as well as the mannosyltransferase activities. Solid ammonium sulfate was added to solubilized microsomal proteins to 60% saturation. After stirring for 4 h at 4 "C, the suspension was centrifuged at 48,000 x g for 30 min. The 69-kDa polypeptide remained in the supernatant fraction, while the 37-kDa polypeptide was recovered in the precipitate. The supernatant was brought up to 80% (NH,,),SO, saturation, stirred at 4 "C for 4 h, and centrifuged as before to collect the precipitated protein. The pellet was dissolved in a small amount of buffer C (50 mM HEPES, pH 8.0, containing 10% glycerol, 5 mM 2-mercaptoethanol, 1 mM benzamidine, and 0.1%. Nonidet P-40 (v/v)) and dialyzed overnight against several changes of the same buffer.

The dialyzed protein, obtained as above, was used in competition experiments, conducted identically as the photolabeling experiments, by including different guanosine-containing sugar nucleotides and other potential competitors of GDP-hexanolamine-[""IIASA. The results of SDS-PAGE of the photolabeled proteins as presented in Fig. 4A shows that, among the different competitors, guanine-containing compounds GTP, GDP, and GMP (lanes 3, 4 , and 1 0 ) were nearly as potent inhibitors as GDP-Man (lane 6); GDP-Glc, GDP-mannuronic acid, guanine, GDP-Fuc, and guanosine (lanes 5, 7, 8, 9, and 13) were less inhibitory, whereas Man-1-P, UDP-Glc, and UDP- Man (lanes 2, 11, and 12) failed to compete with the photoactive reagent for the 69-kDa polypeptide.

In a parallel experiment, the competitors were tested for their effect on the mannosyltransferase activities in the same protein fraction (Fig. 4B). The data are strikingly parallel to the competitive inhibition of insertion of the photoreagent into the 69-kDa polypeptide and lend strong support to the conclu- sion that this polypeptide represents a GDP-Man-requiring mannosyltransferase in the microsomes.

Purification of 69-kDa Pol.ypeptide Representing GDP-Man: Man01 4GlcNAc(31 4GlcNAc-P-P-Do1 Mannosyl- transferase-One hundred grams of the lactating rat mammary tissue was processed to get 200 ml of solubilized microsomal proteins. The solution was brought to 30% (NH,),SO, saturation. After stirring overnight a t 4 "C, the precipitate was removed by centrifugation at 48,000 x g for 30 min. The supernatant was brought to 60% (NH,),SO, saturation and stirred for 4 h at 4 "C. The precipitate representing the 30-60% (NH,),SO, fraction was saved for processing to purify the 37- kDa polypeptide that is also labeled with GDP-hexanolamine- ['"IIASA and is catalytically active for mannosyltransfer from GDP-Man' to a glycolipid. The supernatant from the 60% (NH,),SO, precipitation was raised to 80% saturation with the salt, and the contents were stirred for 6 h at 4 "C. The precipitated protein was separated by centrifugation, dissolved in buffer C, and dialyzed against three changes of the buffer over a period of 24 h.

The dialyzed sample was centrifuged at 10,000 x g for 30 min to remove denatured protein, and the supernatant was loaded on a DEAE-Trisacryl column (1 x 20 cm) equilibrated with buffer C. The column was washed with 3 volumes of the same buffer followed by elution with a linear gradient (0-300 mM) of NaCl in buffer C. Alternate fractions from the column were monitored by the standard assay for mannosyltransferases. The peak fractions of enzymatic activity, eluting between 100 and 150 mM NaCI, were pooled and dialyzed against buffer C for 6 h. The dialyzed pool was applied to a column of concanavalin A-Sepharose (1 x 5 cm) equilibrated with buffer C. The mannosyltransferase activity was recovered in flow-through fractions. It was collected and immediately applied to a column

' M. Ashok, S. Roy, and I. K. Vijay, manuscript in preparation.


FIG. 3. Native PAGE (7.5%) on solubilized microsomal proteins before (upperpanel) and after labeling with the photoactive probe. The gel in the upper pane/ was sectioned for the deter- mination of mannosyltransferase activities, whereas the gel in the lower panel was dried and autoradiographed.

., .- .

of GDP-hexanolamine-Sepharose (0.2 x 7 cm), packed, and equilibrated with buffer C containing 20%. glycerol. The column was washed with 25 volumes of buffer such that no 280 nm absorbing material was present in the flow-through fractions. It was then eluted with the same buffer containing 10 mM GDP-Man. The active fractions were dialyzed against 50 mM HEPES, pH 7.6, containing 0.1% 2-mercaptoethanol, 1 mM EDTA, 1 mM benzamidine, and 150 mM KC1 (buffer D). The dialyzed enzyme was applied to a column of Sephacryl300 (1 x 50 cm) equilibrated with buffer D. The enzyme activity was eluted in a single, symmetrical peak eluting with a molecular mass of 330-360 kDa. The active fractions were pooled and dialyzed in KCI-free buffer D. Glycerol was added to a final concentration of 2576, and the enzyme was aliquoted and stored at -80 for use in characterization and photolabeling studies. A portion of the enzyme eluted from Sephacryl column was dialyzed overnight against 50 mM sodium phosphate, pH 6.8, and used for radioiodination to check for purity. The overall protocol for the purification and the recovery of the mannosyltransferase is summarized in Table I. It should be noted here that from the profile of Dol-P-P-linked [“Clsaccharides synthesized by the microsomes and the solubilized membranes, only -10% of the label is incorporated into the tetrasaccharide. By tha t criterion, the total units representing the c~1,3-mannosyltransferase should be -35,000 in these preparations. The fact that subsequent purification steps do not reflect a significant drop in enzyme units as other mannosyltransferase activities are gradually eliminated implies that there may be an inhibitor of the enzyme that is removed at the ammonium sulfate precipitation step.

There was insufficient enzyme at the end of the final purification step for staining after SDS-PAGE. Therefore, it was decided to iodinate the enzyme and then check for its purity by SDS-PAGE. The iodinated enzyme migrated as a major band of 69 kDa on the gel (Fig. 5, panel A); low levels of slow moving bands near the origin are possibly the products of oxidation/ polymerization of the 69-kDa polypeptide that might have arisen during the iodination reaction. Such by-products are often obtained during iodination of proteins. When the purified

enzyme was photolabeled with GDP-hexanolamine-1125111ASA, i t showed only the 69-kDa polypeptide (Fig. 5, panel B ).

Characterization of the Product of Mannosyltransferase-A scaled up version of the standard incubation was run with the purified mannosyltransferase. The radioactive glycolipid product was hydrolyzed with 0.01 N HCI to obtain I’“C1Man oligosaccharide for analysis. Upon paper chromatography in butanol/pyridine/water, 4:3:4, the oligosaccharide migrated a s Man,GlcNAc, (Fig. 6A). It was resistant to cleavage by endo P-N-acetylglucosaminidase L (Fig. 6B). It was hydrolyzed by endo-p-N-acetylglucosaminidase D and yielded a radioactive fragment that migrated as a trisaccharide when chromatographed on paper in ethyl acetate/pyridine/acetic acidlwater, 5:5:1:3 (Fig. 6C). When digested with jack bean a-mannosidase, [‘“CJMan was released (Fig. 6D).

The enzyme endo L is a chitobiase; Trimble et al. (37) have shown that i t hydrolyzes the bond between 2 N-acetylglu- cosamine residues at the reducing terminus of chitobiosylsac- charides. The enzyme will also hydrolyze, albeit at a somewhat reduced rate, the same linkage in ManGlcNAc,; however, additional mannosylation of this compound totally abolishes sus- ceptibility to cleavage by the enzyme. The enzyme endo D will hydrolyze the chitobiosyl linkage in high mannose oligosaccharides that possess the core structure, provided the mannosyl residue linked 01 3 to the P-mannose in the core portion is not substituted (38). Our earlier investigation had extended the substrate specificity of endo D and shown that the enzyme could hydrolyze Man01 -> 3ManPl 4GlcNAcP1 - > 4GlcNAc lacking the a1 6-linked mannose of the core (29). Taken together with the detailed structural analysis of the Dol-P-P- linked tetrasaccharide for the biosynthesis of glycoproteins in the mammary gland, both in vivo (39) and in vitro (29), the results of the analysis given above indicate that the product of the mannosyltransferase has the structure Manal --> 3Manpl

4GlcNAcPl 4GlcNAc in which the a-linked mannose arises from the transfer of mannose from GDP-Man to the ManPl 4GlcNAcP1 4GlcNAc-P-P-Dol in the acceptor glycolipids added to the incubation mixture. Thus, the mannosyltransferase purified in this investigation represents the en-


FIG. 4. A, inhibition of photoaffinity labeling of the 69-kDa polypeptide by potential competitors of the radioactive probe. The 60-80'2 ammonium sulfate fraction of the solubilized microsomes enriched in thc 69-kDa polypeptide was incubated with 600 nmol of individual inhibitors for 5 min before the addition of 60 nmol of radioactive probe. After photolabeling, the reactions were processed as given under "Experimental Procedures." Lanes 1-14 contained as follows: 1, no inhibitor; 2, mannose-1-P 3, GTP; 4, GDP; 5, GDP- Glc; 6, GDP-Man; 7, GDP-mannuronic acid; 8, guanine; 9, GDP-Fuc; IO, GMP; 11, UDP-Glc; 12, UDP-Man; 13, guanosine; 14, molecular size markers. B, inhibition of mannosyltransferase activity in the presence of different inhibitors. In a set of tubes identical with those given above in A, after incubation with individual inhibitors, GDP-I"C1Man and other constituents of the incubation mixture were added to each tube, and the mannosyltransferase activity was measured by the standard assay procedure.

B 5,000

4,000

3,000

5 0 a

2,000

1,000

0 1 2 3 4 5 6 7 8. 9 1 0 1 1 1 2 1 3

TABLE I Purification of ul,3-mannosyltransferase from rat mammary gland

See the text for experimental details.

Purification Total protein Total units" Specific activity -Fold steps (X1000) (x1000) purification Yield

r/, w Microsomes 95 364 3.8 1 100 Solubilized microsomes 44 323 7.3 1.9 89 (NH,),SO, precipitate 12 309 25.8 6.7 85 DEAE-Trisacryl 1.34 287 214 56 79 Concanavalin A-Sepharose 0.186 253 1360 335 69 GDP-hexanolamine Sepharose 0.004 242 60,500 15,792 66 Sephacryl S-300 NDh 229 ND ND 63

" A unit of enzyme activity is defined as the incorporation of 1000 cpm of [IIClMan from GDP-['IC]Man into productis) under the standard conditions of assay.

ND, not determined.

zyme that transfers the first a-linked mannose residue during obtained at the final step of purification was not enough for the biosynthesis of Dol-P-P-linked Glc,Man,GlcNAc, for pro- injecting into the animals to raise the antibodies. Also, as tein N-glycosylation. shown in Fig. 5,panel A, after radioiodination, this preparation

Monospecific Polyclonal Antibodies against Mannosyl- of the enzyme showed minor slow moving bands that either transferuse-An SDS-PAGE analysis of the enzyme eluted from resulted from oxidation-oligomerization of the 69-kDa polypep- a GDP-hexanolamine-Sepharose column indicated the pres- tide during iodination or represented impurities that might ence of several minor bands besides the 69-kDa polypeptide. still be present in the final preparation. Regardless, it was These minor bands could be representing other mannosyltrans- deemed unsuitable to use either of the enzymes a t these two ferases that might also bind the affinity matrix. The enzyme stages of purification to raise antibodies. To overcome this dif-


A kDa -

69'"

FIG. 5. SDSPAGE (10%) of the mannosyltransferase. The active fractions eluting from the Sephacryl S-300 column were pooled and dialyzed. An aliquot of the pool was iodinated with Na**'I while another aliquot was photolabeled with the radioactive probe. m e r SDS-PAGE of the samples, the gel was dried and autoradiographed. Panel A, three lanes of iodinated enzyme; panel B , three lanes of photolabeled enzyme.

ficulty, as given under "Experimental Procedures," the enzyme eluting from the affbity column was subjected to SDS-PAGE and separately also photolabeled with GDP-hexanolamine- ['2511ASA and electrophoresed alongside. The protein band run- ning parallel to the photolabeled polypeptide, as identified by Ponceau S staining on nitrocellulose paper, was cut out and used to raise the antibodies in the rabbit by standard immuni- zation procedure.

The Nonidet P-40-solubilized microsomal proteins were analyzed for their reactivity to the partially purified IgG fraction of the antiserum on a Western blot. As shown in the Fig. 7, the antibodies recognize a 69-kDa polypeptide among these proteins, indicating their monospecificity (lune B ) . The preimmune serum did not recognize any polypeptide (lane A). Incu- bation of the antibodies with the solubilized rat mammary microsomes inhibited the mannosyltransferase activity; however, even at the highest concentration (antibody/solubilized microsomal protein ratio of 5:1), the inhibition was less than 25%. This is explicable when one considers that the antibodies are directed only toward the 69-kDa mannosyltransferase, whereas the solubilized microsomes have all the mannosyltransferases that utilize GDP-Man as substrate. The solubilized microsomes were subjected to ammonium sulfate precipitation to obtain the proteins precipitating between 60 and 80% saturation. After dialysis, this fraction was tested for inhibition by the partially purified antibodies. As shown in Fig. 8, even this fraction enriched with al,3-mannosyltransferase showed less than 50% inhibition. Thus, the supernatant after antibody precipitation of the solubilized microsomal proteins or 69-kDa polypeptide-enriched proteins may still have other mannosyltransferases that incorporate mannose from GDP-Man into the acceptors. To establish this point experimentally, the standard

250 1

200

150 I a

l o o

50

250

200

2 150 0

loo

so

1 Endo D

1

C

I a 0

1 10 20 30 40

Distance moved (cm) FIG. 6. Chromatogram of the structural studies performed on

[Man-"Cl oligosaccharide derived from mild acid hydrolysis of the mannosyltransferase product. A, migration of [Man-'4Cloligo- saccharide; B, digestion of the oligosaccharide with endo L; C, digestion of the labeled oligosaccharide with endo D; D, digestion of the oligosaccharide with jack bean a-mannosidase. Paper chromatograms were run either in butanovpyridinelwater, 4:3:4 (panels A and B ) , or in ethyl acetate/pyridine/acetic aciawater, 5:5:1:3, for 24 and 18 h, respectively. Arrows 1 5 indicate the migration of the standards: 1, mannose; 2, GlcNA%; 3, ManGlcNAc,; 4, Man,GlcNAc,; 5, Man,GlcNAc,.


A B kDa -200

-46

-30

FIG. 7. Western blot of the mannosyltransferase. The solubilized microsomal proteins from the rat mammary gland were subjected to 10%- SDS-PAGE, transferred onto nitrocellulose paper, and probed with: A , preimmune serum (1500 dilution); B, 1500 dilution of the antiserum. The position of the 69-kDa polypeptide is shown by the arrow- head.

120 I 1

0 1 2 3 4 5

Antiserdsolubilized microsomal enzyme ( W N ) Preimmune sera Antisera - .-*-.

FIG. 8. Immunoinhibition of mannosyltransferase activity with antibodies. The proteins obtained in the 6040% ammonium sulfate fraction of the solubilized microsomes were dialyzed and incubated with different amounts of partially purified I g G fraction of the antiserum or preimmune serum. After precipitation with Protein A- agarose, the enzyme activity was assayed in the supernatant.

mannosyltransferase activity reaction was conducted with the supernatant obtained from the immunoprecipitation of the 69- kDa enzyme from solubilized microsomes. Care was taken to use an antibody/solubilized microsomal protein ratio of 3:l to minimize any nonspecific precipitation of other mannosyltransferases in the immune complex. The [Man-'4Clglycolipid products were hydrolyzed with 0.01 N HCI to release the water- soluble oligosaccharides. The latter were then analyzed by paper chromatography. The results presented in Fig. 9 show that the antibody removes the mannosyltransferase that gives the Man,GlcNAc, product while the other mannosyltransferases are still present in the supernatant. These data further define the specificity of the antibody.

Properties of the Mannosyltransferase-The pH optimum of the purified mannosyltransferase was found to be between 7.4

800 0""""""""""""""""""""""

6

600

400 0

200

~'I"""""""""""""'""'"""""'* 10 20 30 40 5( Distance moved (cm)

FIG. 9. Mannosyltransferase activity in the supernatant from the immunoprecipitated solubilized rat mammary microsomes. The supernatant obtained after immunoprecipitation with the partially purified IgGs of the preimmune serum (panel A ) and immune serum (panel B ) was used as a source of mannosyltransferase activities in standard assay. The products of the reaction were processed to obtain mild acid-released oligosaccharides which were chromatographed on

the migration position of the standards: 3, ManGlcNAc,; 4, paper in butanoYpyridine/water, 4:3:4, for 24 h. Arrows correspond to

Man,GlcNAc,; 5, Man,GlcNAc,; 6, Man,GlcNAc,; 7, Man,GlcNAc,.

and 7.8, consistent with our earlier observation that the biosynthesis of dolichol-P-P-oligosaccharides up to Man,GlcNAc,- P-P-Dol by the solubilized proteins of bovine mammary microsomes show a wide range of pH optimum between 6.8 and 7.6 (31). Also, the enzyme required a divalent cation such as Mg2' and Mn2+ for its optimal activity. Earlier we had shown that biosynthesis of intermediates up to Man,GlcNAc,-P-P-Dol by the microsomal glycosyltransferases in the bovine mammary gland was stimulated by divalent metal ions, notably Mg2' and Mn"; however, the elongation of Man,GlcNAc,-P-P-Dol all the way up to Man,GlcNAc,-P-P-Dol did not require these ions. Rather, the inclusion of Mn2+ or Cu2+ at 10 mM concentration was inhibitory for the elongation reactions (31). Sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide were potent inhibitors of the mannosyltransferase. In the case of p-chloromercuribenzoate, this inhibition could be partially re- versed by dithiothreitol (data not shown). The sugar nucleotides, nucleotides, and other inhibitors of the photoaffhity labeling reaction in Fig. 4A were inhibitory to the purified enzyme similar to the effect shown for the enzyme in solubilized microsomes in Fig. 4B (data not shown). The apparent K,,, of the enzyme for GDP-Man was found to be -4 PM. Due to the lack of availability of pure ManPl- 4GlcNAcPl-+ 4GlcNAc-P-P-Dol, the K,, of the enzyme for the acceptor substrate could not be determined. Among the several phospholipids with different polar head groups, viz. phosphatidylglycerol, -ethanolamine, and -choline, phosphatidylcholine showed an -2-fold stimula- tory effect on the enzyme activity.

Munnosyltransferuse of the Dolichol Cycle 11335

DISCUSSION

This study was undertaken in an attempt to purify, charac- terize, and obtain a molecular probe against one or more mannosyltransferases of the dolichol cycle that are involved in the assembly of the oligosaccharide precursor, Glc,Man,GlcNAc,, for protein N-glycosylation in the mammary gland. Photoaffin- ity labeling has served as an excellent tool in identifying the structure-function relationships of protein-ligand interactions for numerous biological investigations. An important caution in such studies is to ensure the specificity of the photolysis-de- pendent insertion of the highly reactive free radical of the ligand analog. Considering that 5-azid0-UDP-[fl-~~PlUDP-Glc had allowed us to successfully identify the UDP-G1c:Dol-P glucosyltransferase in the rat mammary gland (19) and that GDP- hexanolamine-[’2511ASA was able to inactivate the GDP-Fuc: nLcOse,Cer al,3-fucosyltransferase in a photolysis-based manner which could be protected by GDP-Fuc added prior to photolysis (271, it appeared an attractive proposition to try this approach to identify the mannosyltransferases of the dolichol cycle in the mammary gland. I t was hypothesized that as a substrate analog of GDP-Man, GDP-hexan~lamine-[’~~I]ASA would be able to gain entry into the active site of one or more mannosyltransferases and tag the same. The specificity of insertion of the analog could be examined by competition experiments in which guanylyl derivatives would inhibit photolabeling. The results of this study bear out the validity of the hypothesis. GDP-hexan~lamine-[’~~I]ASA was specifically able to label two polypeptides of 69 and 37 kDa among the detergent-solubilized proteins of the rat mammary microsomes.

While the mannosyltransferases, like other enzymes of the dolichol cycle that have been studied so far, are extremely labile and rapidly lose activity after solubilization of the microsomes with detergents, we were able to stabilize these activities suf- ficiently on the basis of our earlier work (31), so that attempts could be made to obtain one or more of these enzymes to a high degree of purity. It remained to be shown that the photolabeled polypeptides represented mannosyltransferases, or at least their catalytic subunits, rather than GDP-Man-binding proteins, although at least until now, no such proteins or trans- porters of this nucleotide sugar have been identified in the ER. The possibility of the 69- and 37-kDa as GDP- or GMP-binding proteins also could not be ruled out. Native gel electrophoresis followed by an immediate analysis for mannosyltransferase activities among the resolved proteins revealed that indeed two of the peaks of enzyme activities coincided with the photolabeled polypeptides.

A number of glycosyltransferases residing in the Golgi complex have been purified and characterized at both biochemical and the molecular biological level (40-67). The centerpiece in the purification protocols of most of these enzymes has been one or more chromatographic steps involving a sugar nucleotide or its analog as an affinity ligand (earlier work reviewed in Ref. 68). After differentially precipitating the 69- and 37-kDa polypeptides, that were susceptible to photolabeling by GDP- hexan~lamine-[’~~I]ASA with (NH&SO,, we attempted the purification of the 69-kDa polypeptide with GDP-hexanolamine- Sepharose as the affinity chromatographic matrix. We were able to obtain this polypeptide to a high level of purity, if not homogeneity. The enzymatic activity of the polypeptide was shown to represent GDP-Man:Manfll + 4GlcNAcfl1 + 4Glc- NAc-P-P-Dol mannosyltransferase and the product characterized as Mana l - 3Manf l l4 4GlcNAcfll-j 4GlcNAc-P-P-Dol. This represents the enzyme that transfers the first a-mannosy1 residue during the biosynthesis of the oligosaccharide, Glc,Man,GlcNAc,. For simplicity here we will refer to the enzyme as al,3-mannosyltransferase. Even though the enzyme

activity eluted as 330-360 M , protein from the Sephacryl S-300 column under conditions that would minimize the protein ag- gregation, from the limited data, it cannot be stated definitively whether the enzyme is a multisubunit protein. The observation that the 69-kDa polypeptide representing the enzyme remains with the stripped microsomes indicates that it is an integral protein within the ER membranes.

Several polypeptides smaller than 69 kDa also eluted from the GDP-hexanolamine-Sepharose after the application of 10 mM GDP-Man to the column. These did not label with GDP- hexanolamine-[’2511ASA (not shown). Whether they represent low levels of other GDP-mannose-requiring mannosyltransferases, breakdown products of the 69-kDa polypeptide or other nonspecifically bound proteins, cannot be stated definitively.

As a molecular probe, a highly monospecific polyclonal antibody against the al,3-mannosyltransferase was raised in the rabbit. This antibody also specifically immunoinhibited the enzyme activity while other mannosyltransferases remained un- affected. It is rather significant since 6 mannosyltransferases of the dolichol cycle use GDP-Man as the donor substrate, and, except for GDP-Man:Dol-P mannosyltransferase, all the other 5 use Dol-P-P-linked saccharides that differ from one another by a single mannosyl residue as acceptors, raising the possibility of a certain level of relatedness or conservation in the structural architecture of their substrate binding or active sites.

As mentioned earlier, a diverse array of post-ER glycosyltransferases of the secretory pathway have been purified and, in a number of cases, localized in different subcompartments of the Golgi complex through isopycnic gradient sedimentation andor immunocytochemical analysis. Many of these enzymes have been cloned and investigated for their regulation. Still other glycosyltransferases, whose products are expressed at the plasma membrane, have been characterized through a molecular approach by gene transfer into a recipient cell, in what may be termed as “cloning by panning“ (reviewed in Ref. 41). Strategies were developed in which the cDNA or the gene of the glycosyltransferase was transferred and expressed in the recipient cell without the need to first purify the enzyme. The products of these enzymes are expressed on the cell surface. An appropriate reagent among the multitude of antibodies and lectins specific for the oligosaccharide products of different glycosyltransferases could be coated on the cell culture dishes to “pan” for the positive cells or, alternatively, separate the positive recipient cells by fluorescent cell sorting and obtain the clone for characterization. Unfortunately, the enzymes of the dolichol cycle do not express their products on the cell surface and cannot be cloned by the panning approach.

Utilizing mutagenization with [3H]mannose suicide and se- lection, Robbins and collaborators (69-75) have isolated a number of mutants of S. cereuisiae. These mutants have lesions in mannosyltransferases of the dolichol cycle; molecular cloning of several of these enzymes in the yeast has been elucidated. None of the up to 10 mannosyltransferases of the dolichol cycle have been obtained in a homogeneous form or molecular tools developed against the same in any animal system. Because of the lack of a rigorous biochemical and molecular-biological characterization of these enzymes, details for the regulation of protein N-glycosylation in hormonally responsive tissues remain unde- fined. The purification, characterization, and the development of a highly specific polyclonal antibody against the a1,3-mannosyltransferase, as described in this study, provides the first successful effort toward achieving this goal. These results also point to the powerful potential of photoaffinity labeling in identifying and characterizing biological macromolecules that are otherwise difficult to obtain. Our current experiments are de- signed to follow this approach for identifying and purifying additional glycosyltransferases of the dolichol cycle, so that


studies can be undertaken to delineate the regulation of protein N-glycosylation during the hormonally modulated growth and differentiation of the mammary gland. This tissue synthesizes and secretes massive amounts of some of the well-characterized proteins in milk. Among these proteins, the iron transporter, transferrin, and a-lactalbumin, the specifier subunit of the lac- tose synthase complex, are asparagine-linked glycoproteins. As a tissue that is intensely modulated by a variety of hormones during its ontogeny and possesses enormous secretory capacity, the mammary gland offers unlimited potential as a bioreactor for the synthesis and secretion of biomedically significant glycoproteins (76).

Acknowledgment-We thank Margaret Kempf for dedicated secretar- ial assistance.

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THE JOVRNAL UF CHE41fSTRY Vol. 269, No. 15, Issue ofApril ... · G-10 (PD 10 column, Pharmacia) under red light. The purity of the '251-labeled product was checked by thin layer chromatography