M o d A: A post-translational mutation affecting phosphorylated and sulfated glycopeptides in Dictyostelium discoideum Hudson H. Freeze and Arnold L. Miller

Dept. of Neurosciences, School of Medicine, University of California, San Diego, LaJolla, CA 92093, U.S.A.

Summary The Mod A mutation in Dictyostelium discoideum results in a post-translational modification which reduces the activity and electrophoretic mobility of a group of lysosomal glycoproteins. To determine whether this mutation might affect protein bound oligosaccharides, metabolically labeled [2] 3H-mannose glycopeptides were isolated from wild-type (AX3) and mutant cells (M31) of Dictyostelium discoideum. A group of large, negatively charged glycopeptides are significantly depleted in strain M31 compared to AX3. Cells of each strain double labeled with 3H-mannose and 35SO4 or 32[904 showed that the large, negatively charged glycopeptides of AX3 contain both sulfate and phosphate while those of M31 are depleted in these groups. The kinetics of 35SO4 release from the glycopeptides of each strain suggested that both contained similar sulfated sugar(s), but that M31 glycopeptides contained three-fold less than those of AX3. Acid hydrolysis of 3 2 p o 4 containing 3H-mannose glycopeptides showed the presence of 3H-mannose-6-32-Pphosphate in the AX3 hydrolysates while the glycopeptides of M31 contain only 15% as much mannose-6phosphate as those of AX3.

Introduction The recessive mutation, Mod A, in strain M31 of

Dictyostelium discoideum ( D. discoideum) results in partial deficiencies in the activities of a-D-mannosidase, /3-D-glucosidase and N-acetyl /3-D-glucosaminidase (1). These glycosidases together with c~-D-glucosidase, whose activity is unaffected in the mutant strain, are also electrophoretically less negative when compared to the enzymes from wildtype strain AX3 (2). These lysosomal enzymes do not share common polypeptides, but contain a common antigenic component composed in part of carbohydrate (3). The results suggest that the mutation in M31 results from an altered post-translational modification which is shared by the affected lysosomal hydrolases. In order to explore whether protein bound carbohydrate was altered by the Mod A mutation, we examined metabolically

labeled glycoproteins from both strains. Our results indicate that a class of glycopeptides in M31 has altered size, charge and lectin interaction properties compared to those of AX3. Futhermore, the decreased charge of the M31 glycopeptides correlated with a decrease in mannose-6-phosphate and sulfate content of these molecules, A preliminary report of this work has already been published(4 ).

Materials and methods Strains AX3 is an axenically growing wild-type strain as is M31 which was derived from AX3 by N-methyl-N: nitro-N nitrosoguanidine mutagenesis (5). Its isolation and genetic characterization have been reported (2).

Molecular and Cellular Biochemistry 35, 17-27 (1980). 0300-8177/81/0351 0017/$02.20. 9 1981 Martinus Nijhoff/Dr. W. J u n k Publishers b.v., The Hague. Printed in The Netherlands.

18

Growth and Labeling of Cells Cells were grown in 5 to 20 ml of HL-5 medium (6) with 0.5 to 1.0 #Ci/m12114C]-1eucine 354 m C i / m mole and 5 u C i / m l D[2-3H] mannose (2 C i / m mole) (all from Amersham) or in D[2-3H]-man nose alone. Experiments were also performed using 5 # C i / m l of either D-[6-3H]-glucosamine (38 Ci/ mmole) or D[1-3H] galactose (22 Ci/mmole) alone. Experiments involving double labels were carried out with 5 # C i / m l D-[2-3H]-mannose and 5-10 /~Ci/ml of either Na235SO4, (655 m C i / m m o l e ) or 10 /~Ci/ml of Na2H32PO4 (200 m C i / m M ) purchased from New England Nuclear. Experiments involving 3 2 p o 4 w e r e performed in HL-5 medium substituting 2-(N-morpholino) ethane sulfonic acid (MES) for phosphate buffer (7). All labeling was carried out for 24 hr and cells were harvested at a density of 5-8 X 106 cells/ml.

Preparation of cell extracts and glycopeptides Washed cells (1-2 X 108) were suspended in 0.15 M Tris-HC1 buffer pH 7.5 containing 0.002 M CaC12 and 0.02% (w/v) NaN 3 at 1 X 108 cells/ml and lysed with Triton X-100 at 0.1% (v/v). The lysate was digested with 1 mg of pronase (Calbiochem) at 42 ~ for 24 hr and after pH readjustment another 1 mg of pronase was added and the incubation continued for another 24 hr. The samples were heated at 100 ~ for 10 min and then centrifuged. The supernatant was loaded onto a 45 X 1.5 cm column of Biogel P-2 and eluted with 0.05 M a m m o n i u m acetate buffer pH 6.5. An aliquot of each fraction was counted to determine the location of the labeled glycopeptides. The 3H-mannose labeled glycopeptides which eluted in the void volume, were pooled, lyophilized and used for further experiments. Pronase digestion was effective in reducing the content of t4C-leucine or I4Camino acid mixture to near background levels in the Vo of P-2 columns. In all cases the ratio of i4C-amino acid to 3H-mannose was the same in the glycopeptides of AX3 and M31. Cells labeled with 32PO4 and 3H-mannose were lysed as described above and the lysate centrifuged at 120,000 X g for 90 min. The supernatant was then digested with 1 mg RNase, 100 #g DNase and 100

~g c~-amylase (Sigma, St. Louis, Mo.) for 2-4 hr at 25 ~ followed by pronase digestion as described above. Results obtained from glycopeptide preparations following extraction with a 2:1 mixture of ethanol:ether were comparable to those found without the extraction procedure. Incorporation of ~4C-leucine and 3H-mannose into protein was measured following precipitation of 25 #1 of undigested lysate with 10% (w/v) trichloracetic acid (TCA). Precipitates were collected on glass fiber filters, washed with TCA containing 1 m g / m l unlabeled leucine and mannose and counted as described below.

Chromatographic procedures Biogel P-6 Biogel P-6 (200-400 mesh, Biorad) was equilibrated in 0.1 M a m m o n i u m bicarbonate pH 8.0 and packed into 117 X 1 cm silanized glass columns. The columns were eluted with the same buffer containing 0.02% (w/v) NaN 3 at a rate of 0.5 to 0.6 ml per 8-12 min fractions. The V o and V i were determined with bovine serum albumin and ~4Cmafmose respectively. Columns were calibrated using a series of ~4C and 3H-acetic anhydride labeled ovalbumin glycopeptides kindly supplied by Dr. James R. Etchison. Molecular weights of the Dictyostelium discoideum glycopeptides were estimated using the procedure described by Etchison et al. (8) Each figure comparing AX3 and M31 glycopeptides shows an elution profile normalized to the same total 3H-mannose counts.

DEA E sephadex D E A E Sephadex (Pharmacia) was equilibrated in 0.01 M a m m o n i u m bicarbonate buffer pH 8.0 and packed into 10 X 0.7 cm silanized glass columns. Samples were applied in this buffer and eluted with the same buffer containing: 0, 0.05, 0.10, 0.20, 0.40 or 1.0 M KCI in a stepwise fashion. ~4C-acetic anhydride labeled ovalbumin glycopeptide (14C-Ac-Asn N-acetylglucosamine 2 Mannose 6) was added to each sample prior to c h r o m a t o g r a p h y as an internal standard.

19

Concanavalin A-sepharose column chromatography Concanavalin A-Sepharose was equilibrated in 0.01 M Tris-HC1 buffer pH 7.5 with 0.02% (w/v) NaN 3 and poured into silanized Pasteur pipets with plugged with glass wool. Samples of 3H-mannose labeled glycopeptides dissolved in 20 to 100 /zl of equilibration buffer were added to the columns followed by 3 ml wash with equilibration buffer and 3 to 5 ml of equilibration buffer containing 0.2 M c~-methylmannoside. Fractions of 0.5 ml were collected and 0.05 - 0.25 ml aliquots from each fraction were counted. Recovery of AX3 and M31 glycopeptides was 90-95%. Glycopeptide fractions eluted from D E A E Sephadex columns were dialyzed for six hr against 100 volumes of water, lyophilized and subsequently redissolved in 200 #1 of equilibration buffer prior to loading onto the Con A-Sepharose columns. Elutions of these columns were carried out as described above. Control experiments using 3Hmannose labeled D. discoideum glycopeptides showed no loss of sample dialyzed for 6 hr.

Preparation of fetuin glycopeptides Crystalline fetuin was digested with pronase as previously described and the resulting glycopeptides were isolated following column chromatography on Biogel P-2. Carbohydrate and amino acid analysis agreed with values reported in the literature for the asparagine linked glycopeptide (9). The glycopeptides were then labeled with ~4C-acetic anhydride (10) and separated from excess label by chromatography on Biogel P-2.

NaN 3. After 18 hr at 37 ~ C the samples were heated at 100 ~ for 5 min prior to column chromotography on DEAE-Sephadex.

Alkaline phosphatase A 100 #1 aliquot of E. coli alkaline phosphatase (20 U/rag) (Boehringer Mannheim, Indianapolis, Ind.) was dialyzed against 1 liter of 0.01 M a m m o nium bicarbonate pH 8, containing 1 mM MgC12 for 28 hr at 2 ~C. Following dialysis 25 #1 containing about 0.3 U of enzyme activity was added to 50 #1 of M31 or AX3 3H-mannose labeled glycopeptides (10-20,000 cpm) and 25 #1 of 0.1 M a m m o n i u m bicarbonate buffer pH 8 containing 4 / m M MgC12. These samples and the appropriate controls lacking alkaline phosphatase were incubated for 14 hr at 37 ~ C. The samples were then chromatographed on DEAE-Sephadex.

Arylsulfatase A 100 #l aliquot of Arylsufatase from Helix pomatia (5 U/rag, Boehringer-Mannheim) was dialyzed against 1 liter of 0.01 a m m o n i u m acetate buffer pH 6.5 for 28 hr at 2 ~ to remove sulfate. Sampies containing 50 #1 of AX3 or M31 3Hmannose labeled glycopeptides (10-20,000 cpm) were mixed with 25 #1 of 0.1 M a m m o n i u m acetate pH 6.5 and 25 #1 of dialyzed enzyme preparation containing about 0.5 U of arylsulfatase. These samples and the appropriate controls without arylsulfatase were incubated at 37 ~ C for 14 hr. The samples were then chromatographed on DEAESephadex columns.

Enzymatic treatments

Analysis of sulfated and phosphorylated glycopeptides

Neuraminidase treatment

Acid hydrolysis

Samples of AX3 and M31 glycopeptides containing 10-20,000 cpm 3H-ma.nnose or 5,000 cpm of 3H-labeled fetuin glycopeptides dissolved in 50 #1 of water were incubated either in the presence or absence of 0.7 units/ml of C. perfringens Type VI neuraminidase (specific activity of 2 u/rag from Sigma Chemical Co.) and with 50 #1 of 0.1 M sodium acetate buffer pH 5.0 containing 0.04% w / v

Total acid hydrolysis of glycopeptides labeled with 3H-mannose and 35SO4 was performed in 4N HCi for 4 hr and 35SO4 was measured as described below. Glycopeptides containing 3H-mannose3 2 p o 4 w e r e hydrolyzed for 4 hr in 2 N HC1 to generate the sugar phosphates as described by Thieme and Ballou (11).

20

Kinetics of 35S04 releasefrom glycopeptides Procedure used was similar to that described by Margolis and Margolis (12). Samples containing 200 #1 of AX3 or M31 glycopeptides labeled with 3H-mannose and 35804 were mixed with 200 #1 of 0.5 N HC1 and incubated at 100 ~ for up to 250 rain. At each time point, a 20 #1 aliquot was removed and neutralized. Two hundred microliters o f 2 M KC1 containing 100 #g of Na2SO 4 was added to the glycopeptide samples followed by the addition of 300 #g of BaC12 in 150 gl. Barium sulfate was sedimented by centrifugation and the supernatant was removed and counted. Results are expressed as percent of 35804 remaining in solution normalized to free 3H-mannose. Inclusion of 1M KCI prevents artifactual binding of the charged, unhydrolyzed 3H-mannose glycopeptides to the BaSO4 pellet.

Electrophoresis of glycopeptides AX3 and M31 glycopeptides which contained 3H-mannose were pooled from appropriate portions of the Biogel P-6 columns, lyophilized and spotted on cellulose acetate strips. Glycopeptides were subjected to electrophoresis for 40 rain at 16 mA in 1M formic acid at pH 1.92. Glycopeptides of fetuin and ovalbumin labeled with 3H acetic anhydride were run as controls. Strips were then cut into 0.5 em sections eluted with 0.5 ml of 0.1 N HC1 and counted as described. Recovery of 3sS and 3H-was the same on these runs.

35S04 and

Thin layer chromatography of glycopeptide hydrolysis products Glycopeptide fractions from AX3 and M31 containing 3H-mannose and 3 2 p o 4 w e r e hydrolyzed as described above and spotted on 20 X 20 cm cellulose thin layer c h r o m a t o g r a p h y plates which were developed in solvent A: isopropanol/pyridine/acetic acid/water, 8:8:1:4 (13). Ten micrograms of mannose and mannose-6-phosphate were either included as standards in the samples or run adjacent to them. Preliminary runs showed that this solvent system could effectively resolve mannose-6-phosphate, galactose 6-phosphate and glucose-6-phosphate. Sugars were visualized by alkaline silver nitrate staining (14). Lanes containing hydrolyzed glycopeptides were marked at 1 cm

intervals and scraped into scintillation vials containing 0.5 ml of 0.1 N HC1 and sonicated for l0 sec. Scintillation fluid was added and the vials were counted as described below. In some experiments the mannose-6-phosphate region of the above chromatograms was dried, and rechromatographed on another T L C plate, which was developed in Solvent B: butanol/acetic acid/1 N NH4OH , 2:3:1 (15). Prior to thin layer chromatography some of these samples were treated with 0.1U of alkaline phosphatase for 16 hr which was prepared as described above. 3H-mannose release was quantitated following separation on T L C plates in solvent system A.

Counting of radioactivity Radioactive samples were counted in 5 ml of 33% solution of Triton X-100 in toluene containing 4 gm of 2, 5 diphenyloxazole (PPO) and 5.4 mg (1,4-bis 2-5(-phenyloxazole) Benzene ( P o P o P ) per 1 on a Beckman LS8100. All counts are corrected for background and quench. Experiments using double labels were corrected for overlapping counts.

Results

Cells were grown in the presence of ~4C-leucine and either 3H-mannose, 3H-galactose or 3H-glucosamine for 2-7 hr and the trichloracetic acid precipitable counts were measured along with total cellular protein. The results showed that the relative rates and total amounts of ~H-sugars and ~4Cleucine incorporation were the same in AX3 and M31 normalized to protein. The ratio of each 3Hsugar to ~4C-leucine was the same in both strains and indicated that there was no preferential loss of any of these sugars.

Size of glyeopeptides Figure 1 shows the elution profiles of 3H-mannose labeled glycopeptides applied to a Biogel P-6 column. The profile is divided into regions I, II and III of approximate average molecular weights 3800, 2800, and 2300, respectively, based on a comparison with standard glycopeptides. As discussed below, the glycopeptides of Region I are highly charged and the molecular weight estimate for them

21

I 8

II

II

III

J

-

: "'~

I0

,""

6

AX3

......... M31

~" i ~ ~"

9 ' AX3 ...... M31

x

'o x

6

;i

i

O.O5M

0.I0/r

Jl

0.20M

0.40M~

1.0M

Z Z

9z --"..

I0

i

i

20

30

r

40

FRACTION

Fig. 1. Biogel P-6 Elution Profile of 3H-mannose Labeled Glycopeptides from Strains AX3 and M31. Cells were labeled with [2] 3H-mannose and the glycopeptides prepared as decribed in Methods. The glycopeptides were eluted from the 117 X 0.5 cm Biogel P-6 column as described. Results are normalized to the same total 3H-mannose count. Arrow denotes position of ]4C-acetic anhydride labeled ovalbumin glycopeptide: asparagine N-acetylglucosamine2 mannose6 (]4C-AcAsn GluNac2Man~) M.W. 1550. Void column (Vo) is at fraction 8. Regions l, II, lIl are average molecular weights of 3800, 2800, 2300 respectively as determined by standard glycopeptides.

may be in error due to the Well-known charge exclusion effects of Bioge] P-6,(9). Therefore, the differences between Region I and others may be due to size a n d / o r charge. The glycopeptide profile M31 is considerably different than that of AX3. The apparently high molecular weight species of Region ! seen in wildtype is severely reduced in the mutant while there is an increase in the proportion of apparently lower molecular weight glycopeptides of Regions II and III from M31. However, these differences may be due, in part, to differences in charge rather than size. This shift involves a b o u t 30% of the total 3H-mannose. Similar experiments using 3H-galactose and 3H-glucosamine in place of ; H - m a n n o s e resulted in elution patterns for M31 glycopeptides similar to those found for 3H-mannose in Fig. 1, i.e. reduction in the amount of glycopeptides from Region I and increases in Regions II and III. In order to compare charge properties of 3H-mannose containing glycopeptides obtained from AX3 and M31, they were chromatographed on D E A E Sephadex columns and eluted with increasing concentrations of KC1 (Fig. 2). About 30% of the total cell AX3 glycopeptides were eluted by 0.4M KC1

5

I0

15

20

FRACTION

Fig. 2. D E A L Sephadex Elution Profile of 3H-mannose Labeled Glycopeptides from strains AX3 andM31. Glycopeptides were prepared as in Fig. I and normalized to same number of 3H counts for each strain. Arrows denote beginning of elution at indicated concentration KCI (M).

while only about 3% of the M31 glycopeptides were eluted at this KC1 concentration. Instead these glycopeptides appear to elute primarily at 0.1 M KC1. Glycopeptides in Regions I, II and II! of Fig. 1 were ~ooled separately and applied to D E A E Sephadex columns. The AX3 glycopeptides from Region I elute at 0.2 M and 0.4 M KC1 while glycopeptides from Regions II and III elute at 0, 0.05 and 0.10 M KC1. Thus, in M31, the apparently large and most negatively charged glycopeptides are reduced when compared to those of AXY Glycopeptides prepared from ovalbumin and fetuin labeled with ~4C-acetic anhydride were chromatographed on similar D E A E - S e p h a d e x columns. The ovalbumin glycopeptides have one negative charge per glycopeptide and elute with 0.05 M KC1 while the large glycopeptides of fetuin have three to four negative charges per glycopeptide and elute with 0.! and 0.2 M KC1. Thus, by comparison, the glycopeptides of AX3 which elute at 0.4 and 1.0 M KCI are more negatively charged than those which contain two to three residues of sialic acid per chain (9). Neuraminidase treatment of total AX3 glycopeptides prior to chromatography on D E A E - S e p h a d e x produced only minor changes in the elution profile of AX3 compared to untreated samples, and did not result in an elution profile resembling that found for M31 glycopeptides seen on DEAE-Sephadex. Neuraminidase treatment of

22 ~4C-labeled glycopeptides of fetuin followed by D E A E - S e p h a d e x chromatography resulted in a shift in the elution of these glycopeptides from 0.1 to 0.2 M KC1 to primarily 0.05 M KC1.

)

AX3

Lectin binding Concanavalin A was used as a probe to explore other differences which might exist between glycopeptides from AX3 and M31.3H-mannose labeled glycopeptides were chromatographed on columns of Concanavalin A-Sepharose 4B and eluted with 0.2 M c~-methylmannoside as described in the Methods section. Seventy three percent of the M31 glycopeptides bind to Con A-Sepharose 4B compared to 60% of AX3 glycopeptides. Thus, it appears that the Mod A mutation may affect the availability, number or orientation of a-mannose or a-gliacose residues in some, but not all, of the affected glycopeptides from M31. Studies using AX3 glycopeptides fractionated on D E A E Sephadex (as in Fig. 2) showed that the highly charged species are bound least well to ihe Con A-Sepharose (60% unbound) while those species eluting with lower salt are essentially all bound. Thus, it appears that a greater proportion of M31 glycopeptides bind to Con A-Sepharose because the amount of highly charged species is less in this strain. We examined the possibility that a reduction of sulfate or phosphate groups in M31 might acount for the altered behavior of M31 glycopeptides on DEAE-Sephadex.

Sulfated glycopeptides AX3 and M31 glycopeptides labeled with 3H-mannose and 3sSO 4 were analyzed on Biogel P-6 columns (Fig. 3). Panel A shows that AX3 glycopeptides from Region I (as described in Fig. 1) contain 35SO4 and that M31 glycopeptides (Panel B) contains 3-fold less 35SO4 normalized to the same total 3H-mannose. The elution position of the residual 35SO4 found in M31 corresponds only to the trailing edge of the 35SO4 peak seen for the AX3 glycopeptides. Acid hydrolysis of AX3 and M31 glycopeptides followed by barium precipitation showed that 98% of the 35S is in 3sSO4. Cysteine and methionine do not precipitate under these conditions (16). Electrophoresis of 35SO4-3H-mannose labeled

b x

E ~,~

A

2

s

.....r.4 .'i

-g-

~.

"o

0

3

B

M31

Z Z

6

x

6

10

20

30

40

FRACTION

Fig. 3. Biogel P-6 Elution Profile of Glycopeptides of AX3 and M31 Labeled with 3sSO 4 and 3H-mannose. Cells were labeled with 3sSO 4 and 3H-mannose for 24 hr and glycopeptides prepared as in Fig. t. Results of AX3 (Panel A) and M31 (Panel B) were normalized to same total number of 3H counts. Vo at Fraction 6.

glycopeptides from AX3 indicated that 90% of the 35SO4 counts co-migrated with about 18% of the 3H-mannose as a series of peaks considerably more negative than the asparagine linked oligosaccharides of fetuin. Nearly all of the 3H-mannose of glycopeptides from M31 ran considerably less negative than those of fetuin. The residual 35SO4 present in the M31 glycopeptides also co-migrated with about 18% of the 3H-mannose. Kinetics of hydrolysis of sugar sulfates to free sulfate and the appropriate sugar provide preliminary structural information about the sugar-sulfate linkage. Sulfate bound to sugars through esters derived from equitorial, axial, and primary hydroxyl groups have half-lives of 0.1 to 0.4 hr, 1 to 1.5 hr and 1.5 to 2.4 hr respectively in 0.25 N HC1 at 100 ~ (17). Furthermore, the presence of more than one type of linkage in an oligosaccharide can be seen as a bi-phasic kinetic curve (17). Samples of AX3 and M31 glycopeptides were

23

I00

AX3

A

8

60 6 40 4

2

x

20

[

o

co

i

M31

en

=.

o

?

I00

I

I

80

I

I

B

7

~3 8 " x

B

6 60

6 i

4 40

1

20

.,.., I0

20

30

40

FRACTION

1

Fig.5. Biogel P-6 Elution Profile of AX3 and M31 Glycopeptides

i T I M E (hrs)

Fig. 4. K i n e t i c s o f 35So4 R e l e a s e f r o m A X 3 a n d M 3 1 G l y c o p e p -

tides. Glycopeptides of AX3 and M31 labeled with 35SO4 and 3H-mannose (Fig. 3) were separately pooled lyophilized and hydrolyzed in 0.25 N HCI at 100~ for various times. The percent of 35SO4 released from the glycopeptides was determined following barium chloride precipitation as described in Methods. Panel A: AX3 9 9 fractions 13 20 z~---~ fractions 21 25 Panel B: M31 D-- 121 fractions 16 21 m---m fractions 22-28

p o o l e d f r o m c o l u m n effluents s h o w n in Fig 3: P a n el A p o o l of A X 3 f r a c t i o n s 13-20, and 21-25; P a n e l B p o o l of M31 fractions 16-21 a n d 22-28. T h e l y o p h i l i z e d samples were h y d r o l y z e d in 0.25 N HC1 at 100 ~ C for v a r i o u s times. F i g u r e 4, P a n e l A shows that b o t h pools of A X 3 glycopeptides have a halflife o f 210 min and a p p e a r to c o n t a i n only one c o m p o n e n t . P an el B shows that M31 pool 16-21 has a half-life of 208 m i n and also a p p e a r s to c o n t a i n only a single c o m p o n e n t . M31 p o o l 22-28, however, a p p e a r s to c o n t a i n two compo'nents, one with a half-life o f 210 min and a n o t h e r m i n o r c o m p o n e n t with a h a l f life of less t h a n 30 min. T h e s e results

Labeled with 32po4 and 3H-mannose. Cells were labeled and the glycopeptides were prepared as described in Methods. Results of AX3 (Panel A) and M31 (Panel B) were normalized to the same number of 3H-mannose counts. The Vo is at fraction 8 and arrow denotes position of ovalhumin glycopeptide M.W. 1550.

indicate that both A X 3 and M31 glycopeptides c o n t a i n the same or very similar types of sulfated entitibs and that M31 contains 3-fold less of this charged c o m p o n e n t . We have not yet identified the sulfate c o n t a i n i n g sugars in A X 3 and M31 glycopeptides.

Phosphorylated glycopeptides A X 3 and M31 g l y co p ep t i d es labeled with 32p0 4 and 3 H - m a n n o s e were c h r o m a t o g r a p h e d on Biogel P-6 (Fig. 5). Panel A (AX3) shows that the 3 H - m a n n o s e and 32po 4 peaks are c o i n c i d e n t in R e g i o n I. T h e a m o u n t of 32po 4 f o u n d in the g l y c o p e p t i d e f r a c t i o n s of M31 is r ed u ced a b o u t 5-fold c o m p a r e d to AX3. Th e void v o l u m e fractions of A X 3 and M31 also c o n t a i n s s o m e 32po 4 w h i ch m ay be due to residual nucleic acids.

24

Table l. Ten to twenty thousand cpm of ~H-mannose labeled glycopeptides of AX3 and M31 were digested with 0.3 units of alkaline phosphatase or 0.5 units of arylsulfatase for 14 hr at 37~. Controls were similarly incubated in the absence of the enzymes. The samples were chromatographed on columns of DEAE-Sephadex and eluted with the KC1 concentrations indicated. The percent of total 3H-mannose eluted at each step was calculated and the _+ difference of each fraction was compared to the untreated sample. This result was expressed as a _+ percentage. A % a of ~H-mannose in each fraction

DEAE fraction Wash

0.05 M KCI 0.1 0.2 0.4 1.0

AX3 Phosphatase

Sulfatase

M31 Phosphatase

Sulfatase

(18) b (18) (11) (15) (28) (10)

0 +24 +29 6 27 24

(31) + 7 (22) +10 (31) + 8 (7) -26 (3) -41 (6) 60

+ 8 +20 + 8 22 16 50

+ + + +

6 5 6 3 14 6

% 3Hcpm enzyme treated

% 3Hcpm non-treated

a Refers to: _+

XI00 % 3Hcpm non treated

b Numbers in parentheses refer to percent of 3H-mannose counts found in each DEAE fraction prior to enzymatic treatments.

Phosphatase and sulfatase digestion of glycopeptides

is m o r e labile to e n z y m a t i c cleavage than the glycopeptides of AX3.

W h o l e cell g l y c o p e p t i d e labeled with 3 H - m a nnose were i n c u b a t e d with either alkaline p h o s p h a tase, arylsulfatase or no enzyme. T h e r e a c t i o n m i x t u r e was then c h r o m a t o g r a p h e d on D E A E S e p h a d e x , to d e t e r m i n e w h e t h e r these e n z y m a t i c t r e a t m e n t s wo u l d m o d i f y the elution profiles of the g l y c o p e p t i d e s on these c o l u m n s (Table 1). E a c h n u m b e r shows the p e r c e n t change of the 3 H - m a n nose c o u n t s r e c o v e r e d in each f r a c t i o n c o m p a r e d to u n t r e a t e d sample. As an e x a m p l e , if a f r a c t i o n c o n t a i n e d 20% of the total g ly c o p e p t id e s p r i o r to t r e a t m e n t and 10% f o l l o w i n g t r e a t m e n t with one of the e n z y m e s the c h a n g e noted on T a b l e 1 w o u ld be 50%. Th es e d a t a show that A X 3 g l y c o p e p t i d e s are insensitive to alkaline p h o s p h a t a s e t r e a t m e n t , but are m o r e sensitive to sulfatase since a b o u t 25% o f 0.4 M an d !.0 M KC1 species elute at m u c h lo w er ionic strengths f o l l o w i n g sulfatase t r e a t m e n t . Glyc o p e p t i d e s o f M31 are c o n s i d e r a b l y m o r e sensitive to p h o s p h a t a s e t h a n those of AX3. T h e s e results a p p e a r to i n d i c a t e that, while M31 g l y c o p e p t i d e s have decreased a m o u n t s of p h o s p h a t e , the residual

Mannose-6-phosphate T h i n layer c h r o m a t o g r a p h y of acid hydrolysates f r o m the void v o l u m e , R e g i o n s I and II of AX3 and M31 profiles illustrated in Figure 5 show that A X 3 R e g i o n I had 8 to 10% of the 3 H - m a n n o s e and 70% of the 3 2 p 0 4 a s a c o i n c i d e n t peak having the same R f as m a n n o s e - 6 - p h o s p h a t e (Fig. 6). N ei t h er of the pools f r o m the void v o l u m e s of these c o l u m n s showed such a peak. A l t h o u g h g l y c o p e p t i d e s f r o m a p o o l o f R e g i o n s I and II of M31 s h o w e d 3 H - m a n nose and 32po 4 near the a p p r o p r i a t e Rf for m a n nose-6-PO4, the peaks were not c o i n c i d e n t as sh o w n in figure 6. It is possible that a small a m o u n t o f m a n nose-6-PO4 could be present in M31, and that it may be obscured by o t h er labeled, c o - m i g r a t i n g c o m p o n e n t s . T o investigate this possibility, a n o t h e r p r e p a r a t i o n o f 3 H - m a n n o s e and 32po 4 g l y c o p e p tides f r o m A X 3 and M31 R e g i o n s I an d I plus II, respectively were h y d r o l y z e d and d e v e l o p e d by thin layer c h r o m a t o g r a p h y using solvent A. T h e m a n -

25 '

i

A

2o

,

m

4Q

10

.

O

5

10

15

20

FRACTION

Fig. 6. Thin Layer Chromatography of Hydrolyzed AX3 and

M31 Glycopeptides Labeled with 32po4 and 3H-mannose. Glycopeptides containing 32po4 and 3H-mannose were hydrolyzed, dried and spotted on cellulose thin layer plates and developed in solvent A as described in Methods. Regionsof 1cm were scraped, eluted and counted for radioactivity. Arrow denotes position of mannose-6-phosphate. Mannose is found at fractions 13 and 14. Panel A: AX3 Region 1 (Fig. 1). Panel B: M31 Regions 1 and I1 (Fig. 1). nose-6-phosphate region was eluted and spotted on a second TLC plate which was developed in solvent B together with mannose 6-PO 4 and mannose standards. The AX3 glycopeptides showed coincidence of 3H-mannose and 3 2 p o 4 at the Rf of mannose-6-PO4. The M31 sample also showed a 3H-mannose peak at the Rf of mannose-6-Po4, but there was no coincidence of 32po 4. Alkaline phosphatase treatment of both AX3 and M31 hydrolysates prior to chromatography in solvent A showed an 88% conversion of AX3 3H-mannose-6-32po 4 to 3H-mannose while only 13% of the corresponding sample from M31 was converted to 3H-mannose. We conclude that if there is mannose-6-PO4 in the M31 glycopeptides that it comprises less than 15% of that present in AX3 glycopeptides.

Discussion

The Mod A mutation lowers the specific activity and electrophoretic mobility of a group of lysosomal enzymes. These carbohydrate containing enzymes do not share a c o m m o n polypeptide, but they do share a common antigen which is comprised in part of carbohydrate(3). These findings suggested that a loss of a charged carbohydrate group could

account for the electrophoretic differences seen in these enzymes from the mutant strain. Results presented in this report show that glycopeptides prepared from lysates of M31 mutant cells have altered size, charge and lectin binding properties compared to those prepared from AX3 normal cells. It is likely that these altered properties may reflect structural changes in the enzymes affected by the Mod A mutation. Strain M31 contains a group of glycopeptides which appear smaller and less negatively charged than those from wild-type cells. This group of altered glycopeptides accounts for about 30% of the total 3H-mannose. This may be somewhat less on a mole percent basis since it is likely that the larger glycopeptides contain more mannose. The apparent smaller size of the affected M31 glycopeptides may be due to the loss of component sugars, but results using 3H-mannose, 3H-galactose and 3H-glucosamine indicated that there was not the exclusive loss of any single sugar which would account for the decrease in molecular weight. It is likely that at least some of the decrease in apparent molecular weight of these glycopeptides may be due to the loss of sulfate or phosphate. The Region I glycopeptides may only appear to have a higher molecular weight due to the charge exclusion effects seen on these columns (8). The results of ion-exchange chromatography indicate that the large glycopeptides of AX3 are highly negatively charged and require 0.4 M KC1 for elution. This is analogous to yeast phosphomannans which have a mannose to.phosphate ratio of6:1 (10) and require 0.3 M KC1 for elution from similar columns. Neuraminidase treatment of AX3 glycopeptides failed to convert the normal D E A E elution profile into one similar to that of M31, although there were some minor changes in the profile following digestion. These results suggest that a charged group other than sialic acid is responsible for the difference between M31 and AX3 glycopeptides. These results are consistent with the finding that slime molds seem to lack sialic acid (18). Our results suggest that sulfate a n d / o r phosphate is responsible for this charge difference. Glycopeptides of AX3 contain both sulfate and phosphate as shown by incorporation of 35SO4 and 3 2 p o 4 into peaks coincident with the large, negatively charged ~H-mannose glycopeptides (Figs. 3 and 5). Cells of M31 labeled similar to AX3

26 showed a three-fold decrease in the amount of

35804incorporated and a fivefold decrease in 32p04 incorporation when compared to AXY The structure of the sulfated moiety found in AX3 a n d / o r M31 is unknown, but the kinetics of 35804 released from the glycopeptides of AX3 and M31 suggest the presence of a single kinetic species of a half-life of 210 min in 0.25 N HC1 at 100 ~ M31 also appears to contain another species which comprises no more than 5% of the total 35SO4 in M31, and has a half-life of less than 30 rain. These data suggest that there is a quantitative, but not qualitative change in the type of sugar-sulfate in M31. Previous studies in other systems have shown that galactose-6-sulfate, and N-acetylgalactosamine-6-sulfate occur in sulfated glycoproteins and have half-lives of 100 and 148 rain. respectively under these hydrolysis conditions (12, 17). Since sulfate g r o u p s derived from equitorial or axial hydroxyl groups, N-linked sulfate or tyrosine sulfate have much shorter half-lives, the data presented here suggest that the sulfate may be present in a primary hydroxyl group of a sugar other than galactose, glucose or N-acetylglucosamine (17). The phosphorylated glycopeptides of AX3 are about the same size as those which contain sulfate as determined on Biogel P-6. We do not know whether the same glycopeptides contain both sulfate and phosphate groups or whether there are glycopeptides which contain only one of these charged species. However, it is clear that all of the glycopeptides of Region I must contain a strong anionic group since they elute from D E A E Sephadex at 0.4 M KC1. Hydrolysis of AX3 glycopeptides containing 3H-mannose and 3 2 p o 4 followed by thin-layer chromatography of the products in two different solvent systems showed the presence of mannose-6-phosphate. Although the glycopeptides of M31 showed some 32po 4 in Region I, it was at least six-fold lower than in AX3. Furthermore, there was no 3H-mannose-6-32PO4 which was clearly detectable in thin layer chromatograms. Estimations based on the appearance of 3H-mannose following alkaline phosphatase digestion of 3H-mannose 3 2 p 0 4 suggest that the glycopeptides of M31 contain less than 15% as much mannose-6-phosphate as AXY

Mannose-6-phosphate is a component of yeast mannans (11), and recent reports have chemically demonstrated its presence in several mammalian lysosomal enzymes (19, 20, 21). Mannose-6-phosphate residues in a variety of lysosomal enzymes appear to act as a recognition marker for the receptor-mediated uptake of these enzymes by mammalian fibroblasts (22, 23, 24, 25). The prevalance of mannose-6-phosphate in other glycoproteins is unknown. Our data suggests that a group of Dictyosteliurn glycopeptides contains mannose-6-phosphate while glycopeptides of a mutant strain with altered lysosomal enzymes lack it. I cell disease (ICD) is a human metabolic disorder in which a large number of lysosomal enzymes have altered properties related to their carbohydrate moieties (26). Recent studies have shown (27) that several lysosomal enzymes of I-cell disease fibroblasts lack mannose-6-phosphate. These results and our own suggest that I C D and Mod A are mutations that may be unique to lysosomal enzymes. If this is true, the Mod A mutation may affect only glycopeptides derived from lysosomal enzymes, although it is possible that some of the hydrolases may normally modify other proteins including themselves. Lysosomal enzymes are probably all glycoproteins and they comprise about 2% of the cell protein (28). In Dictyostelium about 10% of the cellular proteins are glycosylated (Freeze, unpublished) and thus, the lysosomal enzymes could comprise 20% of the cellular glycoproteins. The results shown here indicate that 30% of the total 3H-mannose label is affected in M3! compared to AX3, although it is probably a smaller mole percentage, since the large, affected glycopeptides should contain more mannose. Thus, it is reasonable that the glycopeptide pattern shifts seen on Biogel and DEAE-Sephadex for M31 could result from only modification of lysosomal enzymes. Our results suggest that the molecular defect responsible for the aberrant glycopeptides in M31 may lead to modification of particular oligosaccharides which are normally phosphorylated or sulfated in AXY Alternatively, a biosynthetic step involved in phosphorylation a n d / o r sulfation of oligosaccharides may be partially deficient in M31.

27

Acknowledgments The authors wish to thank Dr. W. F. Loomis for use of this laboratory facilities for growth of the strains and Dr. James Etchison for standard glycopeptides and helpful advice. The technical assistance of Mrs. Virginia Tejada in the early portions of this work is gratefully acknowledged. This work was supported by N I H Postdoctoral Fellowship F32JL05652 to H. Freeze and N I H Grant NS12138 and R C D A NS00050 to A. L. Miller.

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l l . Thieme, T. R. and Ballou, C. E., 1971. Biochem. 10: 4121-4129. 12. Margolis, R. K. and Margolis, R. U., 1970. Biochem. 9: 4388 4396. 13. Gorden, H. T., T h o m b u r g , W. and Werum, L., 1956. J. S., 1979. Proc. Natl. Acad. Sci. 76:4322 4326. 14. Treveleyn, W. E., Procter, D. P. and Harrison, J. S., 1950. Nature 166: 444. 15. Satio, H., Yamagata, T. and Suzuki, S., 1968. J. Biol9 Chem. 243:1536 1542. 16. Simpson, D. L., Thorne, D. R. and Lob, H. H., 1976. Biochem. 15:5449 5457. 17. Rees, D. A., 1963. Biochem. J. 88:343 345. 18. Warren, L., 1963. Comp. Biochem.Physiol. 10: 153-171. 19. Distler, J., Hieher, F., Sahagian, G., Schmickel, R. and Jourdian, W., 1979. Proc. Natl. Acad. Sci., 76:4235 4239. 20. Natowicz, M. R., Chi, M. M.-Y., Lowry, O. H. and Sly, W. S., 1979. P.N.A.S. 76:4322 4326. 21. vonFigura, K. and Klein, V., 1979. Eur. J. Biochem. 94: 347 354. 22. Kaplan, A., Achord, D. T., Sly, W. S., 1977. Proc. Natl. Acad. Sci. 74: 2026-2030. 23. Kaplan, A., Fisher, D., Achord, D. and Sly, W. S., 1977. J. Clin. Invest. 60:1088 1093. 24. Sando, G. N. and Neufeld, E. F., 1977. Cell 12:619 627. 25. Ullrich, K., Mersmann, G., Weber, E. and vonFigura, K., 1978. Biochem. J. 170:643 650. 26. Miller, A. L., Freeze, H. and Kress, B. C. I-Cell Disease. In: Lysosomes and Lysosomal Storage Diseases (Lowden J. and Callahan, J. W., Eds.) Raven Press, New York (in press). 27. Neufeld E. F., Hasilik, A. and Rome, L. Vth International Symposium on Glyconjugates (in press). 28. Saier, M. H. and Stiles, C. V., 1975. Molecular Dynamics and Biological Membranes, p. 13.

Received August 27, 1980