~ Pergamon PII: S0965-1748(97)00026-X Insect Biochem. Molec. Biol. Vol. 27, No. 6, pp. 513-521, 1997

© 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain

0965-1748/97 $17.00 + 0.130

Insect Mucin-Type Glycoprotein: Immunodetection of the O-Glycosylated Epitope in Drosophila melanogaster Cells and Tissues A. A. KRAMEROV,*t E. A. MIKHALEVA,t YA. M. ROZOVSKY,t T. V. POCHECHUEVA,t N. A. BA1KOVA,t E. L. ARSENJEVA,t V. A. GVOZDEVt

Received 12 December 1996; revised and accepted 12 March 1997

A mucin-type glycoprotein (GP) from cultured embryonic cells of Drosophila melanogaster was isolated and used to raise monoclonal antibodies (MAbs). Epitope(s) recognized by MAbs were sensitive to the treatment by O-glycanase, which specifically cleaves off O-linked mucin- type Gal(/31,3)GalNAc disaccharide, representing the major part of the carbohydrate moiety of Drosophila GP. Using high-affinity MAbs against carbohydrate epitopes of the Drosophila mucin GP we demonstrated its accumulation in culture medium, as well as in cultured cells, which proved to be regulated by 20-hydroxyecdysone. Mucin GPs carrying Gal(/31,3)GalNAc disaccharide recognized by the MAbs were immunochemically localized in several Drosophila tissues of ectodermal, mesodermal and germ line origin, including epidermal and follicle cells capable of their secretion. © 1997 Elsevier Science Ltd

Drosophila Mucin-type glycoprotcin Monoclonal antibody O-glycanase Tissue localization

INTRODUCTION

A large amount of information accumulated in the past decade has demonstrated that vertebrate glycoconjugates play an important role in such biological processes as cell recognition and adhesion, differentiation and mor- phogenesis (for review see Adams and Watt, 1993). Ver- tebrate mucin glycoproteins (GPs), comprising a distinct group of highly glycosylated secreted or membrane-asso- ciated proteins, proved to be of particular importance not only in performing most of these functions, but also in some other cell surface-associated biological phenomena such as fertilization (Florman and Wasserman, 1985) and carcinogenesis (Devine and McKenzie, 1992). Surpris- ingly, despite the potential interest, this group of GPs remains practically uninvestigated, not only in Droso- phila and other insects, but also in invertebrates as a whole.

*Author for correspondence. Fax: + 7 095 196 0221 ; E-mail: dmga@g- las.apc.org

tlnstitute of Molecular Genetics, Russian Academy of Sciences, Kur- chatov Square 46, Moscow 123182, Russia.

Abbreviations: GP, glycoprotein; MAb, monoclonal antibody; PAb, polyclonal antibody; SDS, sodium dodecylsulphate; BFA, brefeldin A; FCS, fetal calf serum; ECM, extracellular matrix; Gal, D-galac- tose; GalNAc, N-acetyl-D-galactosamine; GIcNAc, N-acetyl-D- glucosamine; Xyl, D-xylose.

It was shown earlier that D. melanogaster embryonic cultured cells synthesized a GP comprising a major frac- tion of the newly formed cellular GPs. Its biosynthesis, as revealed by 3H-GlcNac incorporation in Drosophila cells, was regulated by the molting hormone, 20- hydroxyecdysone (Kramerov et al., 1983). On the basis of the high rate of GlcNAc incorporation and chitinase sensitivity GP was proposed to possess a chitin-like carbohydrate moiety (Kramerov et al., 1986). It was also demonstrated that GP-like molecules with similar bio- chemical and antigenic characteristics were synthesized in cultured cells of other insect species and in Drosophila cells in vivo (Kramerov et al., 1990). Further biochemical studies showed that this GP, enriched in Thr, Ser and Pro, carries numerous O-linked mucin-type GaI(/31,3)GalNAc chains (Kramerov et al., 1996). We propose that this GP, named "mucin-D", can be considered to be the first rep- resentative of mucin-type GPs in invertebrates. The aim of this study was to elucidate the localization of mucin- type GPs in Drosophila cells and tissues, using specific monoclonal antibodies (MAbs) raised to the purified GP from cultured embryonic cells. It was shown that the MAbs which recognize epitope(s) containing the Gal(/31,3)GalNAc disaccharide, revealed localization of the mucin GPs to a variety of tissues of different origin, including germ-line cells.

513

514 A. A. KRAMEROV et al.

MATERIALS AND METHODS

Established cell line Dm (67j25D in Kakpakov et al., 1969) and S-2 (Schneider-2 in Schneider, 1972) of D. melanogaster, were cultured on Schneider medium (Gibco, U.S.A.) in the presence of 5% fetal calf serum (FCS; Gibco) as described previously (Gvozdev and Kakpakov, 1968). The cells were replated on fresh medium every 7 days on reaching a concentration of approximately 10 v cells/ml. 20-Hydroxyecdysone (Serva, Germany) was added at the time of replating (1 ~g/ml). Monensin (Serva) and brefeldin A (BFA; Sigma, U.S.A.) were dissolved in methanol and added at concentration of 10/xg/ml. Cell viability was tested by trypan blue staining.

GPs were detected by 2-day metabolic labelling of l 0 7

cultured insect cells with (1-3H)-GIcN (2-5 Ci/mmol; Amersham, U.K.) in 5 ml of culture medium at a concen- tration of 20-40/xCi/ml. Cells were washed twice with saline solution C-15 (Kramerov et al., 1983), lysed in sample buffer (Laemmli, 1970) containing 2% sodium dodecyl sulphate (SDS), and labelled GPs were separated by SDS-gel electrophoresis in 10% polyacrylamide gel (Laemmli, 1970) or in 10-15% gradient Phast-gels (Pharmacia, Sweden). After fixation in 10% trichlo- roacetic acid and staining with a 0.1% solution of Coom- assie blue R-250 gels were impregnated with 2 M sodium salicylate (Chamberlain, 1979), dried and exposed with Kodak XR-like film at - 60°C.

To isolate GP a modification of the previously described (Kramerov et al., 1986) purification procedure was used. Briefly, after washing 10 9 cultured cells in saline solution C-15 the pelleted cells were lysed in 5 ml of 0.5% (v/v) Triton X-100 in C-15 solution. Cytoplas- mic fraction recovered as supernatant after centrifugation of the crude lysate preparation at 80(0 for 20 rain at 4°C, was further treated with pronase E (5 txg/ml) at 37°C for 2 h. After inactivation of the protease by heating at 100°C for 5 min the sample was precipitated with two volumes of cold acetone (overnight, - 20°C). Partly pur- ified delipidated GP was recovered in the pellet after cen- trifugation at 800g for 10 min at 4°C. An additional step in the purification scheme included electroblotting of a gel in 20 mM Na-borate buffer pH 9.2 for 3 h at 0.4 A after preparative gel-electrophoresis in 9% polyacrylam- ide gel of the partly purified GP preparation. An unusually low efficiency of electrotransfer of GP from the gel to a membrane, compared to contaminating pro- teins, resulted in a high degree of purity of GP molecules residing within the gel. Subsequent extraction of GP from the Coomassie-stained gel bands with 50 mM Tris-HC1 buffer, pH 7.0 containing 0.1% SDS, yielded a purified GP preparation with a recovery of approximately 20- 30% as revealed by immunoblot titration of GP contents in crude extract. The isolated GP was dialysed against water and lyophilized. Protein determination was perfor- med according to the method of Bradford (1976) using a Protein Assay Kit (Bio-Rad, U.S.A.).

For O-glycanase treatment (endo-c~-N-acetylgalactos- aminidase, E.C.3.2.1.97) (Boehringer-Mannheirn, Austria) a purified GP (1-2/xg) or acetone-precipitated crude cell lysate (20 x 106 cells) was dissolved in 0.1 ml of 50 mM Na phosphate buffer, pH 7.0, containing 0.5% Triton X-100 and 0.05% SDS, and 1 mU of the enzyme stock solution ( 10 U/mg) was added. Upon incubation at 37°C for 2-16 h, the digestion products were detected after gel electrophoresis either by fluorography or immu- noblot staining.

For immunoblot detection gels were electrophoret- ically transferred in 20 mM Na-borate buffer, pH 9.2 for l - 3 h at 0 .4A onto nitrocellulose filters BA 85 (Schleicher and Schull, Germany) according to the method of Reiser and Stark (1983). Filters were treated for 2 h at 25°C in 0.05% Tween 20 in Tris-buffered saline (TTBS) to decrease the non-specificity of immunostaining, and were then incubated for 1 h at 25°C with the immune serum diluted 1:500 or a monoclonal antibody diluted 1:1000 in TTBS with 5% FCS. After washing three times in TTBS the filters were treated for l h at 25°C with secondary antibodies against either mouse or rabbit immunoglobulins conjugated with alka- line phosphatase (Sigma) in 1:1000 dilution in TTBS with 5% FCS. The washed filters were stained for 15 rain at 25°C with 5-Br,4-CI-3-indolyl-phosphate (Serva) (0,075mg/ml) and nitro blue tetrazolium tSigma~ (0.05 mg/ml) in 50 mM Tris-HCl, pH 9.5. Semi-quanti- tative analysis of the GP contents per cell was accomplished by its immunoblot titration and visual com- parison of the signal with standard serial dilutions of the cell lysate.

Isolation of specific anti-GP polyclonal antibodies (PAbs) has been described elsewhere (Kramerov et al., 1990). MAbs to a highly purified GP sample were obtained using a short protocol of immunization of Balb/c mice, including a primary and one booster injec- tions into pads of hind legs with GP preparation (5/xg) at 7 day intervals. Lymphocytes from regional lymph nodes were fused by polyethyleneglycoi with myeloma cell line SP2. Hybrid cells were selected on HAT-medium and screened using GP immobilized on nitrocellulose mem- brane. Positive hybridoma cells were cloned by the limit- ing dilution method, and the cloned cells were injected into mice for the production of ascites fluid as a source of MAbs.

Tissue localization of the Drosophila GP was perfor- med using both PAbs and MAbs against GP. The paraffin-embedded thin (6 p~m) sections of larva, pupa and imago fixed in Bouin fixative were stained immu- nohistochemically with primary antibodies (in 1:100 dilution for PAbs and 1:300 for MAbs), and peroxidase- conjugated secondary antibodies (Sigma). Immunoperox- idase staining was revealed after incubation of the slides with 0.02% H202 and 3 mM diaminobenzedene (DAB) (Sigma), followed by enhancement of the precipitated DAB product with 0.1% osmium tetroxide. Preimmune serum and antiserum preadsorbed with the purified GP

DROSOPHILA MUCIN-TYPE GLYCOPROTEIN 515

preparation were used as negative controls yielding no specific staining of tissue sections.

Immunoperoxidase staining of 'whole mount' prep- arations of Drosophila embryos 2-12 h after oviposition was performed after fixation in 4% paraformaldehyde in PBS for 30 min at 25°C (Mitchinson and Sedat, 1983) and peeling off vitelline membrane (Jimenez and Campos-Ortega, 1990), as described for tissue sections. Preparations embedded in Canadian balsam were observed using Nomarski optics.

RESULTS

Characterization of the MAbs to mucin-D

MAbs raised to the purified mucin-D preparation (see Materials and Methods) were shown specifically to recognize a 90 kD fraction in the crude lysate of Dm cultured cells metabolically labelled with 3H-GlcNAc (Fig. 1, lanes 1, 5) when analysed by Western blotting. To evaluate the contribution of mucin-type disaccharide units comprising the majority of the sugar chains of GP (Kramerov et al., 1996) to its recognition by MAbs, GP was degly- cosylated by O-glycanase (endo-t~-N- acetylgalactosaminidase), an enzyme known to liberate Gal(/31,3)GalNAc disaccharide connected to a polypep- tide backbone through GalNac-O-Ser(Thr) links (Umemoto et al., 1977). Digestion of 3H-labelled GP with an apparent molecular mass of 90 kD resulted in splitting off the majority of the labelled sugar precursor (Fig. l(a)). During incubation with the enzyme one can detect a progressive loss of the amount of radioactivity in the GP band, accompanied by a decrease of its apparent molecular mass, which finally reached the average value of approximately 70 kD. It should be noted that such a dramatic loss of the labelled sugar chains after O-glycan- ase treatment is caused by the specific activity of the

enzyme which was free of any detectable protease con- tamination, as revealed in model experiments with sev- eral protein substrates (data not shown). Moreover, a low molecular weight product corresponding to a disacchar- ide fraction recovered after digestion of the purified GP by O-glycanase has recently been identified to be Gal(/31,3)GalNAc (Kramerov et al., 1996), fully in accordance with the known enzyme specificity.

The reactivity of six MAbs with mucin-D digested by O-glycanase decreased in a parallel way to that of the radioactivity of the sugar chains. As shown in Fig. 1 (b), O-glycanase treatment resulted in an entire loss of mucin-D staining by one (MAb A7), indicating that the disaccharide split off by the enzyme constituted an important part of an epitope recognized by the antibody. The O-deglycosylatedfraction with a molecular mass of 70 kD, which still preserved some of the labelled sugar chains, thus lacked the epitope(s) recognized by different MAbs against purified GP. We have recently tested the specificities of three MAbs using model carbohydrate structures conjugated to a polymeric matrix. The data obtained showed that all the antibodies do interact specifically with Gal(/31,3)GalNAc, although with differ- ent affinities (Khramtsova, Kramerov, Mikhaleva, Rozovsky and Bovin, unpublished data). A different pat- tern of immunoblot staining was demonstrated for PAbs against mucin-D. These antibodies recognize different types of determinants, including those of the polypeptide part, as revealed by their reactivity with mucin-D devoid of disaccharide chains removed after O-glycanase treat- ment (Fig. l(c)).

Mucin-D secretion by Drosophila cultured cells

Studies of the mucin-D content changes in the prolifer- ating Dm cell line revealed that the amount of mucin-D per single cell detected by immunoblotting in cell lysates

3H_GIcNAc M A b P A b

O-glycanase - - + - - + - - +

1 2 3 4 5 6 7 8 9 10

.92 .92

.66 .66

.45 .45

.92

.66

a b c

FIGURE 1. Treatment of GP from the crude lysate preparation of 3H-GlcN-labelled Dm cells with O-glycanase (10 mU/ml) at 37°C; (a) fluorography; (b,c) immunoblot staining by MAb A7 and PAbs, respectively. Lanes 1, 5 and 9, no enzyme, incubation 16 h; lanes 2 and 6, 2 h; lanes 3 and 7, 4 h; lanes 4, 8 and 10. 16 h treatment. Molecular weight markers: phosphoryl-

ase b (92 kD); bovine serum albumin (67 kD); ovalbumin (45 kD).

516 A . A . KRAMEROV et al.

increased several-fold gradually during cultivation from day 1 to day 7 (Fig. 2(a)). Mucin-D is accumulated within proliferating Dm cells, whereas no apparent increase in net protein contents per cell was observed, as revealed by protein quantititation by the method of Brad- ford (1976) (not shown). In the Dm culture medium mucin-D could be detected just 1 day after splitting of Dm cell culture. The mucin-D concentration in culture medium changed in a parallel way to its intracellular con- centration, leading to a three- to four-fold increase in mucin-D contents which reached - 1 0 - 1 5 % of its total cellular amount at day 7 (Fig. 2(e)). After treatment of Dm cells with 20-hydroxyecdysone that caused inhibition of cell divisions, the mucin-D amount per cell in either Dm cells (Fig. 2(b)) or culture medium (Fig, 2(f)) remained roughly constant or even slightly decreased, compared to its level on the first day after replating.

A variant form of mucin-D with a molecular mass of 80 kD, coinciding with that of its counterpart from Dro- sophila larval imaginal discs (Mukha et al., 1987), was detected in the Schneider-2 (S-2) cell line, although in an amount several times smaller than mucin-D from the

Dm cell line. The intracellular accumulation could also be detected during the cultivation period of the S-2 cell line (Fig. 2(c)). Upon administration of the hormone to the S-2 cells, which halted cell divisions to the same extent as that of Dm cells, a two- to three-fold increase in mucin-D contents was observed compared to the non- treated cells (Fig. 2(d)). In contrast to Dm cells, no detectable amount of 80 kD GP was found in the culture medium of either control or hormone-treated S-2 cells (data not shown).

To elucidate subcellular localization of mucin-D biosynthesis and intracellular transportation, the pro- cesses underlying its accumulation and secretion by cul- tured D. melanogaster cells, we studied the influence on the mucin-D production of the secretion inhibitors monensin and BFA, which were capable of interfering with Golgi functions (Tartakoff, 1983; Misumi et al., 1986). As revealed by MAb A7 immunoblot staining, in the presence of monensin the mucin-D contents in Dm culture medium at day 7 was reduced to 25-30% of its control value (Fig. 2(e,g)). In the BFA-treated Dm cells the biosynthesis of the mature fully glycosylated mucin-

Dm $2 cell line

20-HYDROXYECDYSONE

1 3 5 7 1 3 5 7 1 3 5 1 3 5 DAYS OF CULTURE

.92

.66

.92

.66

.92

.66

.92

.66

- - - - + MONENSIN

1 2 - - + - -

HORMONE

1 3 5 7 5 7 3 5 7 DAYS OF CULTURE

.92

.66

.92

.66

f g

FIGURE 2. Mucin-D accumulation in Dm cells (a,b), S-2 cells (c,d), and in Dm culture medium (e-g) during cultivation as revealed by MAb A7 staining: (a,c,e) control cells; (b,d,f) 20-hydroxyecdysone-treated cells; (g) monensin-treated cells; (hi 3H-GlcN-labelled mucin-D from control cells (1) and cells treated for 3 days with BFA (2) (0.01 mg/ml). Lanes a and b were loaded with 50,000 cells; lanes c and d with 300,000 cells; and lanes e -g with aliquots of culture medium which corresponded

to 300,000 cells.

DROSOPHILA MUCIN-TYPE GLYCOPROTEIN

D with a molecular mass of 90 kD was totally inhibited, whereas the labelled GIcN was incorporated into a het- erogeneous fraction represented by the smear with mol- ecular masses from 60 to 80 kD at a 10% control rate (Fig. 2(h)). The data demonstrated here in favour of mucin-D secretion by cultured cells were further corrob- orated by immunohistological studies in some tissues.

Mucin GP tissue localization and secretion in vivo

The staining of tissue sections with specific antibodies revealed a complex pattern of tissue localization of the GP, which was practically indistinguishable for PAbs and several MAbs used in this study.

Immunostaining was revealed in a number of larval tissues such as fat body cells, oenocytes and haemocytes, capable of producing and secreting components of extra- cellular matrix (ECM) (Fessler and Fessler, 1989), which clearly demonstrated staining of cytoplasmic vesicles resembling secretion granules (Fig. 3(a,c)). Other cell types known to produce cuticle, epithelial cells of imaginal discs (Fig. 3(d)) and epidermal cells of imago, also proved to be abundant in specifically stained cyto- plasmic granules. In the latter case this stained material was exocytosed and included in the newly formed endo- cuticle (Fig. 3(e)).

An impressive immunoperoxidase staining was revealed in follicle cells of adult ovaries beginning from the early stages of egg chamber development. At stages 7-8 (King, 1970) the immunostained granular material was concentrated in the apical region of follicle ceils fac- ing nurse cells and an oocyte (Fig. 3(g)). Later in egg chamber development, the follicular epithelium secreted the stained granules into nurse cells and, especially, into a growing oocyte (Fig. 3(i)). This process continued up to the moment of the degeneration of the follicle cells (stage 11), thus leading to the accumulation in the oocyte of large amounts of the immunopositive material until transformation into a mature egg, where it was concen- trated to the peripheral region (Fig. 30)). No staining of chorion structures or vitelline membrane was observed. Interestingly, similar localization of specifically stained granular material in the apical part of follicle cells was detected after staining of pupal ovaries from the silk- worm Bombyx mori with MAb A7 (Fig. 3(k)).

Specifically stained cytoplasmic granules were detected in primary spermatocytes of pupal (Fig. 3(1)), as well as of larval and adult testes (not shown). In an early Drosophila embryo, mucin GP was localized by immun- operoxidase staining of whole mount preparation in the cytoplasm of blastodermal (Fig. 3(n,o)) and pole cells (Fig. 3(n,p)), but not in the yolk; it could later be found in ectodermal and haemocyte-like cells (not shown).

Western blot analysis was performed to characterize different tissues with respect to the presence of the pro- tein fractions recognized by MAb A7 and their possible diversity in various types of cells. A common fraction with a molecular mass of approximately 80 kD was found in variable amounts in crude Drosophila tissue

517

extracts from imaginal discs, fat body, testis, embryos and ovary, whereas the latter produced an additional 65 kD fraction (Fig. 4). No clear staining of Western blots was detected for flying muscles and salivary glands (not shown).

DISCUSSION

Mucin-D has recently been characterized as a highly glycosylated protein with the sugar moiety amounting to 40% of its molecular weight. We demonstrated a high level of incorporation of labelled 3H-GlcNAc into the Drosophila GP, 10-30 times higher than that of other sugars (Kramerov et al., 1986), which suggested that it has a chitin-like carbohydrate component. However, further study has revealed that the bulk of the radioac- tivity is recovered in Gal and GalNAc (Kramerov et al., 1996). Obviously, 3H-GlcNAc is rapidly converted into 3H-GalNAc that is finally incorporated into the GP. Chemical analysis of sugar chains liberated after alkaline borohydride treatment of mucin-D revealed GaI(/31,3)GalNAc disaccharide to be the main type of O- linked oligosaccharides (Kramerov et al., 1996). These data, along with a high proportion of Thr, Ser and Pro, allowed us to consider mucin-D as a first member of the mucin GP family in insects.

Probing of the GP under study with MAbs with defined specificities could also be an approach to further the underst.anding of its overall structure and possible functions. There are no data currently available which suggest that mucin-D possesses biochemical features of the proteoglycans, comprising another type of highly O- glycosylated protein; mucin-D is not sensitive to chond- roitinase-ABC or endo-/3-galactosidase splitting off sul- phated glycosaminoglycans (not shown). Mucin-D also does not seem to possess Ser(Thr)-O-Xyl linkages because /3-D-xylosides fail to affect its biosynthesis (Kramerov and Gvozdev, 1993). The results of this study also provide no evidence for the presence in mucin-D of a significant amount of O-glycans, other than those of the mucin type. The O-glycanase treatment of mucin-D abolished its reactivity with all the MAbs tested, indicat- ing that GaI(/31,3)GalNAc disaccharide is a predominant type of antigenic determinant exposed on the surface of the mucin-D molecule. We also failed to detect any sig- nificant interaction of the MAbs with desialylated fetuin, known to possess this type of disaccharide (data not shown). The disaccharide probably only comprises a sub- stantial part of the complex epitope, and full-scale high- affinity interactions with the MAbs need some peptide structures also to be preserved in mucin-D.

The results presented in this paper demonstrate that the mucin-D content in cultured cells is a dynamic characteristic and changes during cultivation. Unlike the majority of cellular proteins with their high turnover rates, mucin-D is accumulated within proliferating cells. This observation corresponds well with our previous data on the unusually long half-life of the newly formed GP

5 1 8 A . A . K R A M E R O V et al.

~ j x-~ ,

FIGURE 3. Mucin localization in D. melanogaster tissues from third instar larval stage (a~t), pupa (1), imago (e-j) and embryo (m-p) revealed by immunoperoxidase staining of thin sections (a-l) or whole mounts (m-p) with MAb A7 (a,c,e,k,l,n-p) and PAb (d,g,i,j); (b,f,h,m) treatment with preimmune serum: (a,b) haemocytes; (c) oenocytes; (d) leg imaginal dies; (e,f) epidermal cells from the abdomen of imago; (g,h) follicle cells at stages 6-7 of egg chamber development: (i) staining of tbllicle cells and oocyte at stages 8-9; (j) mature egg; (k) follicle cells of B. mori pupal ovary; (I) primary spermatocytes in pupal testis. 'Whole mount' staining of Drosophila embryo; (re,n) at the cellular blastoderm stage (stage 5) and (o,p) at the early gastrulation stage (stage 6). Bar equals 10 tzm in (a-l,o,p) and to 50 txm in (m,n). Abbreviations: b, blastodermal cells; c, cuticle; lb, fat body cells; fc, tbllicle cells; m, muscle; o, oocyte; pc, pole cells; sg, salivary gland; s, spermatocytes; t, trophocytes; y, yolk.

Arrows indicate granules of the stained material secreted by epidermal or follicle cells.

in the Dm cell l ine (Kramerov et al., 1983). The newly synthesized GP was local ized within cultured cells, mainly in specific cy toplasmic structures resembl ing secretory vesicles or s torage granules (Kramerov et al., 1986). The presence in the GP structure of covalent ly l inked sulphate groups (Kramerov et al., 1986), which are current ly considered to be specific molecular markers of the secretory pa thway (Friedrich et al., 1988; Huttner, 1988), may also indicate poss ib le mucin-D extracel lular localization.

Using M A b s we demonst ra ted the accumulat ion of mucin-D in culture medium as a result of a secretion process which proceeds at a rather low level (10 -15% of

the total amount) in the insect cultured cells tested. The accumulat ion of mucin-D in the culture medium seems not to be the result of cell death because there was no apparent correlat ion between the amount of mucin-D in the culture med ium and the percentage of dead cells, which usually amounted to no more than 1-5%. More- over, the increased (three to five t imes) rate of Dm cell death caused by 20-hydroxyecdysone was not accompanied by an increase in mucin-D extracel lular accumulat ion. Further evidence is provided by the inhi- bit ion of mucin-D accumulat ion in the culture medium of Dm cells by monensin, which indicates that mucin-D secretion proceeds via complex Golg i -med ia ted g lycosyl -

DROSOPHILA MUCIN-TYPE GLYCOPROTEIN 519

K1/

(k)

FIGURE 3. Contd. See previous page for details.

F------q

ation and transportation pathways (Tartakoff, 1983). A more precise subcellular localization of a Golgi subcom- partment involved in mucin-D glycosylation and matu- ration was achieved by treatment of Dm cells with BFA (Misumi et al., 1986). The data obtained may indicate that only a minor part of the labelled precursor, probably corresponding to N-linked chains and/or initiation steps of O-glycosylation, was incorporated in the Golgi mem- branes themselves, whereas the majority of sugars were attached to a polypeptide at later stages, preceding the formation of secretory vesicles in the trans-Golgi net- work. This is in accordance with the data now available on the variable localization of O-glycosylation in ver- tebrate cells, including the enzymatic activities needed for the initiation of O-glycosylation in late compartments of the intracellular membrane network (Hirshberg and Snider, 1987; Wilson et al., 1993).

There is no clear understanding of the apparent differ- ence in molecular mass values of the GP species from Dm and S-2 cell lines. Two mucins might be thought to

be glycoforms of the same glycosylated protein, similar to those described earlier in the sublines of the Dm cell culture, which differed by their O-linked glycans (Kramerov et al., 1996). However, this does not seem to be the case, for two mucin-D forms carry the same type of O-linked sugar epitope, which comprises the major part of their carbohydrate moiety. Two mucins might be considered to be a single molecular entity or closely related entities sharing the main type of sugar chains and also some polypepetide epitopes recognized by PAbs (not shown). We propose that the apparent difference of the molecular masses of two mucin-D species is caused by the different number of disaccharide chains, although truncation of a polypeptide part in the S-2 cell line can not be excluded.

Mucin-D intracellular accumulation and secretion seem to be generally independent of cell proliferation but are affected in two cell lines, although in different ways, by 20-hydroxyecdysone. The hormone caused an increase in the amount of mucin-D in $2 cells, whereas

520 A.A. KRAMEROV et al.

FIGURE 3. Contd. See page 518 for details.

.92

.66

a b c d e f g

FIGURE 4. lmmunoblot staining with MAb A7 of Drosophila crude tissue extracts from: (a) ovary; (b) 1-8 h embryos; (c) imaginal discs; (d) adult testis; (e) fat body; (f) S-2 cells; (g) Dm cells. Lanes a and b were loaded with 0.1 mg, lanes c--e with 0.4 mg and lanes f and g

0.015 mg of total cell proteins.

its accumulation was prevented by 20-hydroxyecdysone in Dm cells. These cell cultures might originate from embryonic cells with different patterns of physiological response to the hormonal stimuli. It has been demon- strated previously that hormonal regulation of gene expression in some Drosophila tissues may be switched from the positive to the negative mode during develop- ment (Anders and Thummel, 1992).

The data obtained demonstrate an apparent ubiquity of the mucin-type epitope(s) recognized by specific MAbs in Drosophila. GPs carrying this epitope are produced by many tissues: derivatives of both ectodermal (epidermal and imaginal disc cells), mesodermal (haemocytes, oeno- cytes and follicle cells) and germ-line ceils (spermatocytes). The variable tissue localization of mucin-D might be caused by recognition by the anti- bodies of various unrelated proteins carrying epitopes containing GaI(/31,3)GalNAc disaccharide. This proposal could not be excluded for several tissues, including oeno- cytes, haemocytes and epidermal cells, which were not

studied by Western blotting. Nevertheless, the results of immunoblot studies showed the presence of the proteins with a similar molecular mass of 80 kD in tissue extracts tested (tat body, testis, imaginal discs, ovary and embryo). Moreover, our earlier data showed that in vitro cultured Drosophila imaginal discs incorporated 'H- GlcNAc into a GP with a molecular mass of 80 kD and biochemical characteristics identical to those of the GP from cultured embryonic cells, including their isoelectric point values (Mukha et al., 1987). These data indicate that the above tissues express an 80 kD mucin GP which is the in vivo counterpart of the 90 kD mucin-D from the cell culture.

Various GPs sharing an O-glycosylated mucin epitope might be proposed to pertbrm different functions when produced in different cell types. They may be involved in the production of insect cuticle by epidermal and imaginal disc cells, and in ECM formation by haemo- cytes which were shown to produce and secrete such ECM components as collagen type IV (Fessler and Fessler, 1989), laminin (Kusche-Gullberg et al., 1992), and a specific proteoglycan-like GP, papilin (Campbell et al., 1987). All these compounds are constituents of basal membranes, but we failed to detect mucin disac- charide localization in this type of structure.

The most interesting finding was that of the active expression of the mucin-type epitope in Drosophila fol- licle cells. A peculiar feature of immunostaining of the ovary is its concentration in the apical part of lbllicle cells preceding the secretion of GPs into an oocyte during its maturation. Such mucin epitope localization in apical parts of the follicle cells was also detected in the ovary of the silkworm B. mori, thus indicating a possible role played by mucin GPs in specifying certain stages of oocyte development in insects. Numerous partly sialyl- ated GaI-GalNAc chains were supposed to be present in

DROSOPHILA MUCIN-TYPE GLYCOPROTEIN 521

the vitellogenin receptor from the follicle cells of Locusta migratoria (Hafer and Ferenz, 1991), suggesting that together with mucin-D this GP comprises a family of insect mucin-type GPs localized in the follicle cells and probably involved in oogenesis. Their possible contri- butions to some oocyte functions can be motivated by the well-documented roles played by several mucin-like GPs from the oocyte envelope in sperm-egg recognition and fertilization in vertebrates (Florman and Wasserman, 1985; Garbers, 1989).

Our results suggest that the MAbs recognizing the spe- cific mucin-type epitope in a variety of Drosophila tissues reveal mucins implicated in different functions, e.g. participation in cuticle or extracellular matrix forma- tion, or some other functions which are still to be investi- gated.

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Acknowledgements--This work was supported by INTAS grant N 93- 0772 and RFFR grant 97-04-48113.