The Effect of Tunicamycin, an Inhibitor of Protein ...THE JOURNAL OF Bmr.mrc~~ CHEMI~TFLY Vol. 253,...

THE JOURNAL OF Bmr.mrc~~ CHEMI~TFLY Vol. 253, No 7, Issue of April 10, pp. 2348.2355, 1978

Printed m U.S.A.

The Effect of Tunicamycin, an Inhibitor of Protein Glycosylation, on Embryonic Development in the Sea Urchin*

(Received for publication, December 2, 1977)

E. GAYLE SCHNEIDER,$ HANH T. NGUYEN, AND WILLIAM J. LENNARZ

From the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

A transferase that catalyzes the synthesis of N-acetylglucosaminyl(pyro)phosphoryldolichol from LJDP-N-acetylglucosamine and endogenous dolichol phosphate has been found in membranes prepared from embryos of Arhacio punctu/ata. -V-acetylglucosaminyI(pyro)phosphoryldolichol synthesis by this membrane transferase is stimulated 16 fold by exogenous dolichol phosphate. The addition of tunicamycin to isolated membranes results in almost total inhibition of the enzyme. Moreover, membranes isolated from embryos treated in oiuo with tunicamycin exhibit no detectable transferase activity. This antibiotic has no effect, either in vitro or in oiuo, on the enzyme that synthesizes mannosylphosphoryldolichol. When embryos are treated with tunicamycin early in development, e.g. as early as 5 h after fertilization, no morphological effects on development are observed until early gastrula, where development is arrested. Thus, the presence of this drug has no detectable effect on morphogenetic changes involved in the development of the embryo from the l&cell stage to early gastrula. It is only at the gastrula stage, when extensive morphogenetic movements of mesenchymal cells begins, that the effect of prior treatment with tunicamycin becomes apparent.

Treatment of embryos with tunicamycin after gastrulation results in the arrest (or retardation) of spicule formation and arm growth. Spicule formation is temporally correlated with enhanced incorporation of [“Hlglucosamine and Yar+ into macromolecules, and the extent of tunicamycin inhibition of spicule (arm) growth is roughly proportional to the extent of inhibition of [SHlglucosamine incorporation. The experimental results suggest an essential role for glycoproteins synthesized by the lipid-linked pathway at specific stages during embryonic development. The effects of tunicamycin on morphogenesis are discussed with regard

* This work was supported by grants from the National Institutes of Health (HD08357) and The Rockefeller Foundation (GA HS 7512). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a Special Postdoctoral Fellowship from The Rocke- feller Foindation. Piesent address, Laboratory of‘Molecular Aging, Gerontology Research Center, National Institutes of Health, Balti- more City Hospitals, 4940 Eastern Avenue, Baltimore, Maryland 21224.

to the possible production of cell surface and/or extracellu- lar proteins deficient in carbohydrate side chains.

In recent years much interest has been focused on the possible role of glycosyl transferases and glycoproteins in cell- cell interactions (l-4). Certainly one of the most challenging problems in developmental biology today is the mechanism(s) by which embryonic cells migrate and orient themselves in a highly specific, organized manner. Several workers have suggested that embryonic cells may migrate on hyaluronic acid-like matrices (5, 6). Others have shown that collagen- and glycosaminoglycan-containing basal lamina support nu- merous cell interactions (7, 8). Recently a role for cell surface glycosyltransferases in gastrulating chick embryos has been proposed (9, 10).

With regard to the sea urchin, very little is known about

glycosyltransferases or glycoproteins and their possible role in embryonic development. In a previous paper (11) we reported the presence of a galactosyl transferase and a mannosyl transferase in eggs and embryos ofArbacia punctulata. It was suggested that the galactosyltransferase may be of the type that adds distal galactosyl units to glycoproteins, and that the mannosyl transferase may be involved in the lipid-linked pathway found in other systems for the biosynthesis of the oligosaccharide core of N-glycosidically linked glycoproteins (12, 13). Other investigators have reported production of acid mucopolysaccharides (14) and appearance of new cell surface determinants (15) at gastrulation in the sea urchin. However, until recently it has been difficult to investigate the specific role of glycoproteins or glycosaminoglycans in embryonic development. With the recent discovery of the antibiotic tunicamycin (16), which specifically inhibits the synthesis of N-glycosidically linked glycoproteins (17-25), it is now possible to study the possible function(s) of this class of glycoproteins in embryonic development.

We report here on the presence of a tunicamycin-sensitive transferase found in cell-free preparations of embryos of A. punctulata that catalyzes the synthesis of N-acetylglucosaminyl(pyro)phosphoryldolichol from UDP-N-acetylglucosamine and dolichol phosphate. In view of findings in other systems (13, 18) it seems likely that this enzyme is involved in the first step of the lipid-linked pathway for the glycosylation of asparagine-linked glycoproteins (13, 21, 26). The effect of

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Tunicamycin Effects on Embryonic Development

addition of tunicamycin to 16-cell embryos (e.g. 5 h after fertilization) is not manifested until early gastrula, where development is arrested. Treatment of gastrula with tunicamycin blocks transition to the prism stage. Similarly, treatment of prisms with the drug blocks development at the pluteus stage. Parallel labeling experiments at these two later stages of development show that the normal increase in incorporation of labeled glucosamine into macromolecules is

inhibited by tunicamycin. The observations that tunicamycin inhibits GlcNAc-(PI-P-dolichol’ synthesis and causes arrest- ment of development at specific stages suggest an essential role of glycosylation of proteins by the lipid-linked pathway during embryonic development.

EXPERIMENTAL PROCEDURES

Materials-A. punctulata were purchased from either Florida Marine Biological Specimen Co., Panama City, Florida, or Shark River Marine Biological Supply Co., Wall, N.J. The animals were maintained in aquaria at 18” in artificial sea water (Instant Ocean, Aquarium Systems, Inc.). UDP-N-acetyl-[U-14Clglucosamine (300 mCi/mmol) was purchased from Amersham/Searle Corp. [CH,,- “HlThymidine (51.8 Ci/mmol), asCa2+ (23.7 mCi/mg), 16- 3Hlglucosamine (20.7 Ci/mmol), and GDP-[U-14Clmannose (210 mCi/ mmol) were obtained from New England Nuclear Corp. Dolichol phosphate was prepared by Dr. Dorothy Pless, formerly of this laborat,ory, by the method of Wedgwood et al. (27). Tunicamycin was a gift of Dr. G. Tamura, University of Tokyo. All other compounds were obtained from commercial sources unless otherwise noted.

Growth ofA. punctulata Embryos: Treatment with Tunicamycin - Gametes were obtained from animals by electrical shock, fertilized, and grown at a density of approximately 6 x lo” embryos/ml (0.2 to 0.4 mg of protein/ml) in artificial sea water containing gentamycin, as previously described (11). At the hatched blastula stage (or other appropriate time), the embryos were collected, washed, and resuspended in artificial sea water containing 2 rnM Tris/HCl, pH 8.0, at a density of 2.4 x lo5 embryos/ml (12 mg/ml). For embryos younger than hatched blastula, the fertilization envelope was removed by treating the embryos with hatching enzyme obtained from the growth medium of a preparation of hatching embryos as previously described (28). Tunicamycin (1.26 mg/ml in 0.025 M NaOH) was added to one aliquot of embryos to give a final concentration of 12 pg/ml of tunicamycin; to the control embryos, an equal amount of 0.025 M NaOH was added. The two batches of embryos were incubated for 20 min at room temperature with occasional gentle mixing. Each batch was then diluted approximately loo-fold with artificial sea water containing gentamycin to identical final concen- trations of approximately 6 x lo” embryos/ml (0.2 to 0.4 mg of protein/ml) and incubated at 18” with gentle stirring. At various times embryos were removed and observed in the phase contrast microscope to determine the extent of embryonic development. For all such determinations at least 200 embryos were observed.

with 2.5 ml of 0.9Yo saline and then three times with 4 ml each of 0.9% NaCl:methanol (2:l). Labeled lipid in the washed chloroform extract that was labile to mild acid hydrolysis was determined as follows: the chloroform layer was evaporated under N,, and the residue was dissolved in 0.3 ml of tetrahydrofuran:0.5 M HCl (4:l). The mixture was incubated for 2 h at 50”, neutralized with 60 ~1 of 0.5 M NaOH, and the solvent was evaporated under N,. To determine the amount of radioactivity converted into a water-soluble form the hydrolyzed mixture was dissolved in 1.5 ml of chloroform:methanol (2:1), 1 ml of water was added, and the mixture was vortexed thoroughly. The aqueous layer was removed and saved. The organic layer was washed once with 1.5 ml of 33% methanol; the combined aqueous layers were then dried in a scintillation vial and counted in 1.0 ml of H,O plus 10 ml of Hydromix (Yorktown Research) or ACS (Amershamlsearle).

Assay of [3H]Glucosamine, %‘a”+, or [3HlThymidine Incorporation by Embryos-To assay incorporation of [“Hlglucosamine, *Wa’+, or [3HIthymidine in u&o, approximately 3 x 10” embryos (1 to 2 mg of protein) were suspended in a total volume of 0.5 ml of artificial sea water containing 2 &i of [“Hlglucosamine, 1.5 pC!i of Wa’+, or 2 &i of [3Hlthymidine. The embryos were incubated with the appropriate isotope for 0 to 60 min at room temperature with gentle mixing by rotation. [“HlGlucosamine or [3HIthymidine-labeled embryos were sonicated at 0” in 2.5 ml of 5% trichloroacetic acid, and acid-insoluble material was recovered after centrifugation at 10,000 x g. The [“Hlglucosamine- or [:‘Hlthymidine-labeled pellets were washed twice with 5 ml of 5% trichloroacetic acid. The washed pellets were then suspended by sonication in 1 ml of water and counted in 10 ml of scintillation fluid. Wa”+-labeled embryos were washed extensively (approximately eight times) with 5 ml of artificial sea water followed by brief centrifugation at 100 x g. The washed embryo pellet was suspended in 0.7 ml of 90% NCS tissue solubilizer (AmershamiSearle), heated at 70” for several hours, and then counted in 10 ml of Hydromix or ACS scintillation fluid.

Miscellaneous Methods- Respiration was determined by measur- ing the rate of oxygen uptake from the medium with an oxygen electrode. Synthesis of mannosylphosphoryldolichol was measured as previously described (11). Protein was determined by the method of Lowry et al. (29).

RESULTS

Preparation of Membrane Fractions- Embryos were lysed by hy- potonic shock in Tris/EDTA (20 rnM Tris/HCl, pH 7.6, containing 5 rnM EDTA), and membranes were recovered by centrifugation at 100,000 x g, as previously described (11). The membranes were washed once again in Tris/EDTA and then in 0.1 M Tris/HCl (pH 8.5); the final membrane pellet was resuspended in 0.1 M TrislHCi, pH 8.5, to a concentration of 10 mg of protein/ml.

Assay of N-Acetylglucosaminyl Transferuse -N-Acetylglucosami- nyltransferase activity was measured by incorporation of N-acetyl- [‘4C]glucosamine from UDP-N-acetyl [14C]glucosamine into lipids that were labile to mild acid hydrolysis. The assay system consisted of 10 rnM MgCl,, 100 mM Tris/HCl (pH 8.51, 0.1% Triton X-100, 0.10 rnM dolichol phosphate, 16.7 PM UDP-N-acetyl-[‘4Clglucosamine (specific activity = 3.5 x IO5 cpm/nmol), and 150 to 500 pg of enzyme protein in a total volume of 0.1 ml. Dolichol phosphate and Triton X- 100 were added together as an aqueous suspension. After incubation at room temperature 2.5 mg of ovalbumin were added and the reaction was stopped by the addition of 4.5 ml of chloroform:methanol (2:l). The precipitate was removed by centrifuging at 1000 rpm for 15 min and washed once with 1 ml of chloroform:methanol (2:l). The chloroform:methanol (2:l) extracts were combined and washed once

Enzymatic Synthesis of GlcNAc-(P)-P-Dolichol- When crude particulate fractions of Arbacia punctulata embryos are incubated with UDP-N-acetyl-[‘“Clglucosamine in the presence of Mg2+, the major lipid-soluble, 14C-labeled product is a compound that is labile to mild acid hydrolysis. To investigate the possibility that the acid-labile glycolipid was GlcNAc-(PI- P-dolichol, the effect of exogenous dolichol phosphate on its synthesis was investigated. In the presence of dolichol phosphate synthesis of the acid-labile lipid was markedly stimulated (Fig. 1). To confirm that the acid-labile lipid is GlcNAc-

(PI-P-dolichol, it was analyzed by thin layer chromatography on silica gel 60 plates (E. Merck Corp.). After double development with CHCl,:CH,OH:H,O (60:35:6) a single radioactive peak identical in mobility with authentic GlcNAc-(P)-P-dolichol (261, and well separated from authentic /3-GlcNAc- GlcNAc-(P)-P-dolichol, was observed. Greater than 90% of the ‘“C-labeled water-soluble product released upon mild acid

hydrolysis of the lipid was identified as N-acetylglucosamine by paper chromatography in the following solvent systems: 1) butanol:pyridine:water, 60:40:30; and 2) nitromethane: ethanol:acetic acid:boric acid-saturated water, 8:l:l:l. These findings establish that under the assay conditions reported here the major product is GlcNAc-(PI-P-dolichol, not p- GlcNAc-GlcNAc-(P)-P-dolichol.

Properties of Transferuse ~ The transferase catalyzing synthesis of GlcNAc-(P)-P-dolichol exhibits a Mg’ + optimum of 10 mM, a pH optimum of 8.5, a dolichol phosphate optimum of 100 PM, and an apparent K, for UDP-N-acetylglucosamine of 5 PM. Under these assay conditions less than 10% of the sugar nucleotide is degraded by the membrane preparation. The

1 The abbreviation used is: GlcNAc-(PI-P-dolichol, N-acetylglucosaminyl(pyro)phosphoryldolichol.

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2350 Tunicamycin Effects on Embryonic Development

enzyme appears to be inhibited by UMP and is extremely sensitive to Mn’+ and possibly other heavy metal ions. Thor- ough washing of isolated membranes with EDTA (5 mM) is essential for detection of activity. Since tunicamycin has been shown to inhibit the GlcNAc-(PI-P-dolichol synthetase of calf liver (18), chick embryo (17), yeast (191, and hen oviduct (21) membranes, the effect of this antibiotic on the enzyme from A. punctulata membranes was investigated. As shown in Table I, the enzyme in membranes from A. punctulata embryos that catalyzes synthesis of GlcNAc-(PI-P-dolichol is completely inhibited by tunicamycin. In contrast, Man-P-dolichol synthesis by these membranes is unaffected.

Effect of Tunicamycin on Early Embryonic Development- In view of the finding that tunicamycin, which is known to

Mmutes

FIG. 1. Stimulation of GlcNAc-(P)-P-dolichol synthesis by exogenous dolichol phosphate. Particulate enzyme (0.15 mg) from 50-h embryos was incubated with UDP-N-acetyl[W]glucosamine in the presence or absence of dolichol phosphate for the times indicated (see “Experimental Procedures”). The isolated W-labeled lipid was subjected to mild acid hydrolysis (see “Experimental Procedures”) and the released N-acetyl[14Clglucosamine was measured.

inhibit synthesis of N-glycosidically linked glycoproteins, inhibits the in vitro synthesis of GlcNAc-(PI-P-dolichol, it was of interest to see what effect in viva treatment with tunicamycin might have on the development of the early embryo. A series of experiments in which embryos were treated with tunicamycin as early as 5 h or as late as 17.5 h after fertilization revealed that in all cases development was arrested prior to the prism stage. As illustrated in Fig. 2, 16-cell stage embryos denuded of their fertilization envelope by hatching enzyme and then treated with tunicamycin initially develop in a manner identical with untreated, denuded embryos. Both untreated (Fig. 2a) and treated embryos (Fig. 2e) developed cilia and began to swim at the same time (12 h). Observations by phase contrast microscopy revealed little difference in morphology between control (Fig. 2b) and tunicamycin- treated embryos (Fig. 2f, at 24 h. However, it was clear that the treated embryos (Fig. 2, g and h) did not proceed to the prism or pluteus stages (36 and 48 h) observed with the control

TABLE I Effect of tunicamycin on glycosyl transferases of A. punctulata in

vitro

Addition to assay N-acetylglucosaminyl transferase” Mannosyl transferase*

nmollmglh nmollmglh

None 0.043 5.03 Tunicamycin <0.0003 5.17

o Particulate enzyme (0.148 mg) from 50-h embryos was preincubated for 10 min in the complete assay system (see “Experimental Procedures”) without sugar nucleotide in the presence of 1 ~1 of 0.025 M NaOH or 1 ~1 of 0.025 M NaOH containing 1.25 yg of tunicamycin. UDP-N-acetyl[‘“C]glucosamine was then added to start the reaction.

b Particulate enzyme (3 Kg) from 50-h embryos was preincubated for 10 min in the complete assay system without GDP-mannose in the presence of 1 ~1 of 0.025 M NaOH or 1 ~1 of 0.025 M NaOH containing 1.25 pg of tunicamycin. GDP-[Wlmannose was then added to start the reaction.

FIG. 2. Photomicrographs of developing embryos. Embryos were denuded of fertilization envelope with hatching enzyme at 5 h and then allowed to develop with or without pretreatment with tunicamycin. Morphological development of embryos not treated with tunicamycin: (a) 12 h, swimming blastula; (b) 24 h, gastrula; (c) 36 h, prism- early pluteus; (d) 50 h, pluteus. Mor- phological development of embryos pretreated with tunicamycin as described in the text: (e) 12 h; (f, 24 h; (g) 36 h; (h) 50 h. Magnification: x 170.


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Tunicamycin Effects on Embryonic Development 2351

embryos (Fig. 2 c and d). By examination of thick sections of embryos after treatment with tunicamycin at hatched blastula stage (12 h) it was possible to more precisely define the effect of the drug on developmental morphology. It is clear that the first morphologically detectable effect of tunicamycin treatment occurs at 18 h. At this time control embryos are begin- ning to gastrulate and primary mesenchyme cells are visible in the blastocoel (Fig. 3~2). In contrast, tunicamycin-treated embryos exhibit little invagination (Fig. 3~0, although the primary mesenchyme seems to have formed normally. This effect is more pronounced at 21 h (compare Fig. 3, b and e). At 25 h control embryos have completed gastrulation (Fig. 3~1, whereas tunicamycin-treated embryos are only slightly invag- inated and the primary (and possibly secondary) mesenchyme cells are arranged in a single pile on the basal plate (Fig. 3fl. Thus, it seems that in the presence of tunicamycin, the vegetal plate can invaginate only slightly and that the rear- rangement of mesenchyme cells does not occur or is greatly retarded.

FIG. 3. Photomicrographs of thick sections of control embryos and embryos treated with tunicamycin. At the indicated times embryos were fixed in 3% glutaraldehyde in sea water. Embryos were then washed in sea water, fixed in 0~0, for 2 h at 0”, dehydrated with ethanol, and embedded in Epon. Sections of 1 p in thickness were prepared, stained as described elsewhere (30), and photographed in the light microscope. (a) 18 h, (b) 21 h, and (c) 24-h control embryos; (d) 18 h, (e) 21 h, and (f) 24-h embryos8;;tF;t;d with tunicamycin at 12 h. Magnification: a and d, x , , , , andf, x 750.

At least 95% of the embryos that had been treated with tunicamycin at 12 h of age remained viable (as judged by motility) for at least 36 additional h. To determine by more biochemical means that the arrest of development at the early gastrula stage by tunicamycin was not due simply to a nonspecific, toxic effect of the antibiotic, several properties of control and treated embryos were examined. As shown in Fig. 4, treated embryos were observed to consume oxygen at nearly normal rates up to 35 h (early pluteus in controls), despite the fact that their development was blocked at early gastrula. The respiration of treated embryos after 35 h is observed to fall relative to the controls; however, it should be noted that, even after this long period of arrested development, oxygen con- sumption does not fall below the levels observed for gastrula. Fig. 5 illustrates the effect of tunicamycin on [“Hlthymidine incorporation by developing embryos. Embryos were treated with tunicamycin at 12 h, and thymidine uptake into trichloroacetic acid-insoluble material was measured at the times indicated. It can be seen that thymidine uptake by the midgastrula stage (24 h) in treated and untreated embryos is identical, although at later time points the treated embryos take up much less thymidine than control embryos. In any case it is clear that the effect of tunicamycin on morphogenesis precedes its effect on [“Hlthymidine incorporation by at least 6 h.

2- /'

,G ,,Pr E;Pl , L;Pl , , I. *

30 40 50 Age of Embryos (hrs)

FIG. 4. Effect of tunicamycin on respiration. Embryos were treated with tunicamycin at 12 h. Respiration (0, uptake) was measured at the times indicated. O---O, untreated embryos; 0- - -0, tunicamycin-treated embryos. G, gastrula; Pr, prism; E- PL, early pluteus; L-X, late pluteus.

100 - .

90 -

EO-

To 70 - b, ;;60- \

5 50- ‘\ \ : .

r 40- \ \

\ : \ . 5 30- : " :

20- b----o--,

10 - G Pr E-P1 L-P1

I 20 ' ' ' 40 ' 60

Age of Embryos (hrs.)

FIG. 5. Effect of tunicamycin on 13Hlthymidine uptake by developing embryos. Embryos were treated with tunicamycin at 12 h, 13Hlthymidine uptake into trichloroacetic acid-insoluble material was measured as described under “Experimental Procedures.” O-0, untreated embryos; 0- - -0, tunicamycin-treated embryos. G, gastrula; Pr, prism; E-PZ, early pluteus; L-PC, late pluteus.


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The effect of tunicamycin on general protein synthesis was investigated by determining the effect of this antibiotic on the uptake and incorporation of 3H-amino-acids into trichloroacetic acid-insoluble material. When embryos were treated with tunicamycin at 12 h, exogenous amino acid incorporation was depressed by a constant amount (30 to 50% depending on the experiment) from 18 through 48 h. It should be stressed that these results cannot strictly be interpreted to mean that tunicamycin causes a 30 to 50% inhibition of protein synthesis since incorporation of an exogenous precursor does not neces- sarily reflect the rate of protein synthesis (see “Discussion”).

I f the effect of tunicamycin on embryonic development is a direct result of inhibition of protein glycosylation by the lipid- linked pathway, then tunicamycin should inhibit the incorporation of glucosamine into N-glycosidically linked glycoproteins. To investigate this possibility, embryos at various stages of development were incubated for 1 h in the presence of [3Hlglucosamine. Fig. 6 illustrates the incorporation of [3H]glucosamine into trichloroacetic acid-insoluble material in the presence or absence of tunicamycin (added at 12 h). It is clear that only a low level of incorporation is observed in both control and treated embryos until the prism stage. In other experiments, no significant glucosamine incorporation was observed in embryos as young as 12 h (hatched blastula) up through 28 h (late gastrula). It is evident from the results shown in Fig. 6 that a dramatic increase in incorporation of [3H]glucosamine occurs between the prism and the pluteus stages in the untreated embryos (soZid lines). This increase in labeling is completely abolished by addition of tunicamycin to embryos at 12 h (Fig. 6, dashed lines). Moreover, untreated embryos take up 45Ca2+ during the transition from prism to pluteus (see below) but not at earlier stages; pretreatment of embryos with tunicamycin results in complete inhibition of this uptake. However, because the tunicamycin-induced block in development at the early gastrula stage occurs many hours prior to the tunicamycin-sensitive increases in incorporation of glucosamine and Ca*+, it is clear the inhibition of some earlier event must be responsible for the observed block at early gastrula. Clearly, further studies are necessary to define the molecular alterations induced by tunicamycin which lead to the arrest of development at the early gastrula stage.

Effect of Tunicamycin on Late Embryonic Development- Because of the dramatic increase in [3H]glucosamine and

:f ,c$! ,-,P, , ( ..-_-..----a- ..-.._ _.._. _ ___. D

20 40 50 Ago of Embryos (hrsl

3-5 Age of Embryos (hrs.1

FIG. 6. Incorporation of L3Hlglucosamine by A. punctulata em- FIG. 8. Effect of tunicamycin on incorporation of [“Hlglucosamine bryos in the presence (O- - -0) or absence (O----O) oftunicamycin. and +%az+. Embryos were treated with tunicamycin at 46 h as Tunicamycin was added to embryos at 12 h as described under described under “Experimental Procedures.” [3H]Glucosamine and “Experimental Procedures.” Glucosamine incorporation into trichlo- Wa2+ were measured as described under “Experimental Proce- roacetic-acid insoluble material was measured as described under dures.” O-0, O-O, untreated embryos; A- - -A, &--A, “Experimental Procedures.” tunicamycin-treated embryos.

&Ca2+ incorporation at the prism-pluteus stage and the complete inhibition of this incorporation by early pretreatment with tunicamycin (Fig. 61, the effects of tunicamycin on this later stage of development were studied in more detail. Initial experiments revealed that addition of tunicamycin at 27.5 h (i.e. between the gastrula and prism stages) results in nearly total inhibition of PHlglucosamine incorporation. Moreover, the treated embryos failed to develop to the prism stage. As shown in Fig. 7, when tunicamycin was added to embryos at the prism stage (31 h) or the early pluteus stage (35.5 h), [“Hlglucosamine incorporation was again greatly reduced. It

Age of Embryos (hrs)

FIG. ‘7. Effect of tunicamycin on incorporation of [3H]glucosamine by A. punctulata embryos. Embryos were treated with tunicamycin at prism (31 h) or at early pluteus (35.5 h) as described under “Experimental Procedures.” [3HlGlucosamine uptake was measured as described under “Experimental Procedures.” O----O, untreated embryos; 0- - -0, embryos treated with tunicamycin at 35.5 h, a- - -A, embryos treated with tunicamycin at 31 h. Pr, prism; E- PL; early pluteus; M-PL, midpluteus; L-PL, late pluteus.

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FIG. 9. Photomicrographs of embryos treated with tunicamycin at 31 h or 35.5 h and fixed with 3% glutaraldehyde in sea water at 44 h. a, 44-h embryos pretreated with tunicamycin at 31 h; b, 44-h embryos pretreated with tunicamycin at 35.5 h; c, 44-h control embryos.

can be seen that addition of tunicamycin at 31 h completely inhibits glucosamine incorporation, whereas addition of tunicamycin at 35.5 h significantly, but not completely, inhibits incorporation. In a separate experiment, where tunicamycin was added to 46-h embryos (Fig. 8), incorporation of 13H1glucosamine and 45Ca2+ was inhibited 44% and 35%, respectively, by 48 h. Thus, it appears from these later experiments that tunicamycin has a relatively rapid inhibi- tory effect on both [“Hlglucosamine and 45Cas+ incorporation. Enhanced incorporation of 45Ca’+ into an insoluble form at the prism-pluteus transition has previously been shown for at 5 10 15 20 25 30

least one other species of sea urchin, Pseudocentrotus de- Age of Embryos (hours1

pressus; moreover, the incorporated 45ca2+ appears to be concentrated in the spicules (31). It should be noted in Fig. 8

FIG. 10. Effect of tunicamycin treatment of A. punctulata em-

that incorporation of [“Hlglucosamine slightly precedes ‘VaL+ bryos on the level of the transferase that catalyzes synthesis of GlcNAc-(PI-P-dolichol. Embryos were grown and treated with tuni-

uptake. Calcification processes in other systems are known to camycin at 12 h as described under “Experimental Procedures.” At

involve glycosaminoglycans (32-36). It is possible that the various stages of development membrane preparations were made

tunicamycin effect on later development in the sea urchin from aliquots of control (0-O) and tunicamycin-treated

embryo involves inhibition of synthesis of an essential glyco- (O- - -0) embryos. The membranes were then assayed for the dolichol phosphate-stimulated N-acetylglucosaminyltransferase as

protein(s) necessary for skeletal (spicule) formation. That this described under “Experimental Procedures.”

may indeed be the case is strongly suggested by the observa- tion that tunicamycin inhibits arm bud development. As shown in Fig. 9, embryos treated at 31 h (prism) or 35.5 h

incorporation, it is quite possible that the tunicamycin-sensi-

(early pluteus) with tunicamycin and observed at 44 h appear tive, [“Hlglucosamine-labeled macromolecule(s) is highly sul-

to have stunted arms when compared to an untreated control. fated. Preliminary experiments suggest that a large fraction

Preliminary Characterization of [3HIGlucosamine-labeled of the incorporated [:lHlglucosamine-labeled protein is associ-

Product(s) - Strong acid hydrolysis (6 N HCl; 8 hl of the 13Hl- ated with isolated spicules.”

labeled product synthesized during the prism-pluteus stage Effect of Tunicamycin on in Vivo Levels of UDP-N-acetyl-

resulted in release of approximately 60% of the radioactivity glucosamine:Dolichol Phosphate Transferase - If the ob-

as 13Hlglucosamine, 30% as [RHlgalactosamine, and 10% as an served morphological and biochemical effects of tunicamycin

unidentified product. At least 73% of the %H-labeled product is discussed above are due to inhibition of GlcNAc-(P)-P-dolichol

sensitive to pronase digestion, as measured by solubility in synthesis (and, therefore, a blockage in the lipid-linked path-

5% trichloroacetic acid. Mild base hydrolysis (0.1 N NaOH; way for glycoprotein synthesis), the enzyme that synthesizes

37”; 48 h) results in the liberation of only 35% of the radioactiv- this compound should be found to be specifically inhibited in

ity, thus suggesting that less than one-half of this material is vivo by the antibiotic. The level of the transferase catalyzing

0-glycosidically linked to protein. It is, therefore, quite prob- synthesis of GlcNAc-(PI-P-dolichol in A. punctulata embryos

able that the major portion of the [nH]glycosamines are linked as a function of development is shown in Fig. 10. No radical

to protein through an N-glycosidic bond to asparagine. This change in the level of the enzyme during development is

conclusion is consistent with the fact that synthesis of this observed between 10 and 50 h. In contrast, it is clear that the

macromolecule(s) is inhibited by tunicamycin. Chromatogra- transferase cannot be detected in isolated membranes from

phy of a pronase digest of the radioactive macromolecule(s) on embryos treated in vivo with tunicamycin. Thus, if embryos

Sephadex G-50 yields 53% of the radioactivity in the excluded are treated with tunicamycin at 12 h no activity is detectable

volume and 47% as three distinct glycopeptide fractions. in the isolated membranes at 24 h (midgastrula). Other

Chromatography of the excluded fraction from Sephadex G-50 experiments indicate that inactivation is complete within 3 h

on a Dowex l-Cl ion exchange column indicates that the major after in vivo treatment with tunicamycin. In contrast to these

product of pronase digestion is a highly negatively charged findings on the N-acetylglucosaminyltransferase, no inactiva-

molecule. Since in other experiments (data not shown) an tion of the mannosyltransferase is observed.

increase in s5S0,2- incorporation was observed at the prism- pluteus transition concomitant with enhanced lqH]glucosamine

y H. T. Nguyen, G. Hart, and W. J. Lennarz, unpublished observations.


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DISCUSSION

The N-acetylglucosaminyltransferase that catalyzes synthesis of GlcNAc-(P)P-dolichol in membranes of A. punctulata embryos resembles that found in other systems (17-25) with regard to its inhibition both in vitro and in vivo by tunicamy- tin and its stimulation by dolichol phosphate (19, 26). To our knowledge, this report constitutes the first demonstration of this enzyme in an invertebrate. Evans and Bosmann (37) have reported the presence of a number of glycosyltransferases in Lytechinus pictus embryos, including an N-acetylglucosaminyltransferase; however, the [‘“Clhexose-labeled endogenous products insoluble in phosphotungstic acid were not further characterized. The level of N-acetylglucosaminyltransferase, like that of the mannosyltransferase of A. pun&data (ll), does not change appreciably during the course of embryonic development. Preliminary experiments involving assay for N- acetylglucosaminyltransferase at pH 7.6 suggest the formation of both N-acetylglucosaminyl(pyro)phosphoryldolichol (70%,) and N,N’-diacetylchitobiosyl(pyro)phosphoryldolichol (30%). Therefore, it seems likely that the entire lipid-linked pathway for the glycosylation of proteins may be present in A. punctulatu; further experiments are underway to verify this.

(39), cleavage and hatching of embryos are retarded, and dissociation of embryonic cells is observed at the early gastrula stage. These effects were not observed with tunicamycin.

Tunicamycin has been shown to specifically inhibit the synthesis of GlcNAc-(P)-P-dolichol of A. punctulata, both in

vitro and in viuo. If glycosylation of proteins by the lipid- linked pathway is essential for normal embryonic development, tunicamycin should interfere with this development. The results of this study show that, indeed, tunicamycin has a striking effect on development. Treatment of embryos with tunicamycin prior to gastrulation results in the arrest of development at the early gastrula stage, whereas treatment of embryos after gastrulation (i.e. 27.5 h or beyond) results in the arrest (or retardation) of spicule development and arm growth.

Treatment of embryos with the amino acid analog ethionine results in a delayed effect on development (40) which most closely resembles the effect of tunicamycin pretreatment on gastrulation. Very early embryos treated with ethionine were observed to develop normally until the early gastrula stage, at which time there appeared to be a degeneration of the primary mesenchyme (40, 41). In addition, protein, RNA, and DNA synthesis was inhibited approximately 40410, 30%, and lo%, respectively, at the time of the gastrulation block (40).

Thus, it is quite possible that synthesis of abnormal proteins, either with an abnormal amino acid or without a carbohydrate residue(s), may bring about similar effects on embryonic development at the gastrula stage. Of key concern here is whether these observed morphological effects are the result of synthesis of specific defective proteins, perhaps synthesized at or shortly before the gastrula stage, that are crucial to normal gastrulation and development. Indeed, synthesis of acid mucopolysaccharides (14) as well as cell surface determinants (15) has been observed at the time of gastrulation in sea urchin embryos.

The arrest of development at the early gastrula stage of embryos pretreated with tunicamycin clearly represents a delayed effect of this antibiotic on development. Thus, treatment with tunicamycin at either 5 h (16-cell stage) or 12 h (hatched blastula) of development has no detectable morphological effect until gastrulation begins at approximately 18 h. These results strongly suggest that either the synthesis of N- glycosidically linked glycoproteins does not occur in embryos prior to the initiation of gastrulation, or, if such synthesis does occur, it is not essential for development up to this stage.

Embryos deprived of sulfate grow normally only until the mesenchyme blastula stage; these embryos are found to produce primary mesenchyme cells with abnormal surface fea- tures which do not migrate properly (14). Thus, treatment with ethionine or tunicamycin, or sulfate deprivation, all appear to produce similar effects on embryonic development, i.e. they appear to affect the ability of the primary mesen- thyme cells to migrate. It is interesting to speculate that ethionine or tunicamycin treatment, like sulfate deprivation, may interfere with the synthesis of new surface glycoproteins essential for normal cell movements and interactions at gastrulation. Further experiments are now in progress to more clearly define the early tunicamycin effect and to see what relationship, if any, it has to production of sulfated mucopolysaccharides at gastrulation.

The arrest (or retardation) of embryonic development by tunicamycin after gastrulation appears to be related to formation of the skeleton (spicules). Clearly, inhibition of late morphological development by tunicamycin is correlated with an inhibition in [“Hlglucosamine, 15Ca2+, and “Y+O,‘- incorporation into macromolecules. As noted earlier, there is strong evidence implicating all three of these components in assem- bly of skeleton in other systems, and it seems likely that this is also true in the case of the sea urchin. Indeed, the extent of the morphological effect (decreased arm length) is roughly proportional to the extent of inhibition of [“Hlglucosamine incorporation. Hopefully, further studies will define the pre- cise mode of action of tunicamycin in inhibiting this developmental process, as well as its striking, early effect on gastrulation.

Inhibition of gastrulation by pretreatment with tunicamy- tin is not due to a delayed toxic effect on the embryos. The blastulae of treated embryos are motile for many hours after the time at which gastrulation should have occurred. Further- more, embryos treated at 12 h with the antibiotic respire normally until 35 h and exhibit normal levels of thymidine incorporation until at least 24 h. However, protein synthesis as measured by incorporation of labeled amino acids into macromolecules appears to be inhibited by 30 to 50% from 17 h on in embryos treated with tunicamycin at 12 h after fertilization (data not shown). It is important to note, however, that this effect on exogenous amino acid incorporation could be due to increased pool sizes, decreased transport, actual protein synthesis, or a combination of these. It should be emphasized that inhibitors of general protein synthesis such as puromycin and cycloheximide are observed to have early and profound effects on DNA synthesis, cleavage, and respiration in sea urchin embryos (38). These effects were not observed with tunicamycin. Likewise, although treatment of embryos with dactinomycin results in a delayed effect seen at gastrulation manuscript.

Acknowledgments-We thank Doctors D. K. Struck, W. W. Chen, K. K. Kronquist, G. Hart, E. D. Schmell, and W. Kinsey for valuable discussion and suggestions. We are partic- ularly indebted to Mr. G. L. Decker and Dr. W. Kinsey for their efforts on the microscopic and ultrastructural aspects of this study and to Dr. A. Heifetz for thin layer analysis of the glycolipid. We thank Dr. B. Reynafarje for assistance in measurement of respiration of embryos with an oxygen electrode. We thank Ms. A. Fuhr for expert typing of this


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E G Schneider, H T Nguyen and W J Lennarzdevelopment in the sea urchin.

The effect of tunicamycin, an inhibitor of protein glycosylation, on embryonic

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