THE JOURNAL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAOF BIOLOGICAL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBACHEMISTRY zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAQ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 2, Issue of January 14, pp. 146%1476,1994 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAPrinted in U.S.A.

Molecular Cloning and Characterization of the Full-length cDNA Encoding the Developmentally Regulated Lysosomal Enzyme @-Glucosidase in Dictyostelium discoideum*

(Received for publication, July 1, 1993, and in revised form, September 7, 1993)

John Bush, Jan Richardson$, and James CardelliQ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFrom the Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, Louisiana 71130

The developmentally regulated Dictyostelium dis- coideum lysosomal enzyme fl-glucosidase is synthesized as a membrane-associated glycosylated precursor poly- peptide which undergoes at least two proteolytic cleav- age events to generate a soluble mature lysosomally localized protein. To begin to analyze the mechanisms regulating the sorting of this protein and the regulation during development of the expression of the encoding gene, we have cloned and sequenced a 2.6-kilobase (kb) cDNA which contains a complete 2463-nucleotide open reading frame coding for @-glucosidase. Concep- tual translation of this open reading frame predicts a polypeptide similar in molecular mass to the primary translation product of 94 kDa that also contains the same amino acid sequences of two VS protease derived- peptides generated from the purified &glucosidase en- zyme. The D. discoideum enzyme contained regions highly homologous at the amino acid sequence level to both bacterial and fungal &glucosidases, although these regions did not overlap. A potential cleavable signal sequence was also found in the first 21 amino acids followed by a highly polar stretch of 49 amino acids which (based on amino acid sequencing of the mature @-glucosidase) represents a pro region for this protein. This region is similar in location, size, and charge to the D. discoideum a-mannosidase pro-I re- gion (Schatzle, J., Bush, J., and Cardelli, J. (1992) J. Biol. Chem. 267, 4000-4007). Several small hydro- phobic stretches of amino acids were also distributed throughout the protein; however, no obvious trans- membrane region(s) were identified which might ex- plain the observed membrane association of the pre- cursor protein. Finally, Northern blot analysis indi- cated that the gene encoding this enzyme was under developmental regulation. The steady state level of a 2.7-kb &glucosidase mRNA decreased significantly during the aggregation stage of development, from

*This research was supported by National Institutes of Health Grant DK 39232 (to J. C.) and National Science Foundation Grant DCB-9104576 (to J. C.). 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.

The nucleotide sequence(s) reported in this paper zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAhas been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L21014.

$ Current address: Dept. of Physiology, Dartmouth University School of Medicine, Lebanon, NH 03766.

To whom all correspondence should be addressed Dept. of Mi- crobiology and Immunology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130. Tel.: 318-674- 5756; Fax: 318-674-5764.

high levels during growth, and then increased in the form of a larger size 2.8-kb mRNA during the final stages of development.

Mammalian lysosomal enzymes are transported and tar- geted to lysosomes by at least two different mechanisms (reviewed in Kornfeld and Mellman (1989)). The best char- acterized lysosomal enzyme targeting mechanism involves the generation of mannose 6-phosphate residues on the Asn- linked oligosaccharide side chains of lysosomal enzymes. These residues serve as recognition markers for mannose 6- phosphate receptors which mediate transport of lysosomal enzymes to lysosomes. Mannose 6-phosphate receptor-inde- pendent mechanisms for lysosomal/vacuolar targeting in mammalian and yeast systems have also been described (Kornfeld and Mellman, 1989; Klionsky and Emr, 1990).

Dictyostelium discoideum is a haploid eukaryotic organism which appears to use a mannose 6-phosphate receptor-inde- pendent mechanism to sort lysosomal enzymes (for review, see Cardelli (1993)). Because it is amenable to both genetic and biochemical approaches, this organism represents a useful system to study alternative lysosomal targeting mechanisms. D. discoideum cells also can be induced by starvation to undergo a relatively simple developmental cycle (Loomis, 1982). Approximately 6 h after development begins, the cells begin to migrate by chemotactically responding to pulses of CAMP and ultimately form aggregates of 10‘ cells. Aggregates then pass through a number of morphological stages ending in culmination that results in the formation of a mature fruiting body consisting of two types of cells: a stalk of vacuolated cells (stalk cells) which supports a sorus containing dormant spore cells.

Three of the better characterized D. discoideum lysosomal enzymes, a-mannosidase, ,&glucosidase, and acid phosphatase are synthesized on membrane bound polysomes, co-transla- tionally inserted into the rough endoplasmic reticulum, and glycosylated on Asn residues (Cardelli et al., 1987; Cardelli, 1993). During transport of the membrane-associated precur- sor forms of these enzymes, the mannose-rich carbohydrate side chains are sulfated and phosphorylated in the Golgi complex (Mierendorf et al., 1985; Cardelli et al., 1986; Freeze, 1986). Sulfate and perhaps phosphate are not required for correct localization of enzymes (Cardelli et al., 1990a; Bush and Cardelli, 1990; Freeze et al., 1989). During transport to lysosomes, these three enzymes are also subjected to several proteolytic events which generate the soluble mature form of each hydrolase (Mierendorf et al., 1985; Pannell et al., 1985; Cardelli et al., 1986; Bush and Cardelli, 1989). The initial

1468

~ ~ s o s o ~ a l ~ - G l u c o s ~ ~ e : Sequence and ~ e g u ~ t i o n zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1469 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFIG. 1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBARestriction maps of full

length p-glucosidase cDNA and other isolated partial &glucosidase clones. The restriction maps of partial clones and the full-length clone pBG-C and the corresponding ORF (shoded) are shown. The translational start ATG and s t o ~ TAA codon are also designated. The doked line for pBG-16 represents the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAA BG-C length of the non-~-glucosi~se fusion cDNA.

H 0.2kb

cleavage event occurs in a Golgi and/or endosomal compart- ment (Cardelli et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAal., 1990b; Wood and Kaplan, 1988) and may be required for correct localization of enzymes to lyso- somes (Richardson et al., 1988), while final proteolysis occurs within dense acidic lysosomes and is catalyzed by a cysteine proteinase (Cardelli et al., 1989; Richardson et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAaL, 1988). Although the lysosomal and endosomal vesicles are acidic, low pH is not essential for the correct sorting these hydrolases (Cardelli et aL, 1989). The maintenance of acidic vacuolar compartmen~ is, however, required for complete processing of all three lysosomal enzymes. In addition, about 10% of the precursor forms of these enzymes escape cleavage and exit the cell through a constitutive secretory pathway while the mature forms of the enzymes are released from cells in a regulated fashion (Mierendorf et ai., 1985; Pannell et al., 1985; Ebert et al., 1990).

The expression of the two lysosomal enzymes, a-mannosi- dase and @-glucosidase, is regulated during development of D. discoideum (Loomis, 1975). The structural gene encoding a- mannosidase has been cloned (Schatzle et d., 1992) and shown to be regulated at the level of transcription in response to both a secreted protein termed the presta~ation response factor and by starvation (Clarke et aL, 1988, Schatzle et aL, 1991, 1993); however, less is known about the regulation of the expression of the @-glucosidase gene. The rate of &- glucosidase synthesis and the steady state level of enzyme activity increases slightly between 1 and 3 h into development, decreases significantly by the aggregation stage, and then increases steadily from 18 h of development until final for- mation of fruiting bodies (Golumbeski and Dimond, 1987; Coston and Loomis, 1969). The changes in the levels of functional @-glucosidase mRNA correlates with the rate of enzyme synthesis suggesting that expression may be con- trolled at a pretranslational level.

TO begin to determine the molecular factors essential for the targeting of @-glucosidase to lysosomes and the regulation of @-glucosidase expression, the cDNA coding for this protein has been cloned and sequenced. The gene is subject to a complex mode of developmental regulation and the predicted @-glucosidase protein has features shared with another D. discoideum lysosomal enzyme, a-mannosidase.

MATERIALS AND METHODS

Growth and Development of D. discoideum-D. discoideum strains Ax3 (wild-type), Ax4 (wild-type), and Ga2 (null mutant) derived from an Ax3 parental strain (a kind gift of Dr. R. Firtel) were grown axenically in TM medium (Free and Loomis, 1974) at 21 "C in a rotary shaker water bath rotating at 200 rpm or on SM agar medium at 21 "C in association with K l e ~ s ~ l ~ aerogenes. To initiate devef- opment on filters, vegetative amoebae were collected by centri~gation

and resuspended in cold MES'-PDF buffer (8 mM MES, 0.7 mM CaC12, 0.3 mM streptomycin sulfate, pH 6.5). Cells were placed under developmental conditions as previously described (Schatzle et al., 1991).

Purification of (3-Glucosidase-Four liters of Ax3 cells at 1-2 X lo7 cells/ml were separated from the medium by centrifugation at 3000 X g for 10 min. The supernatant, which contained 90% of the total &glucosidase enzyme activity, was chromatographed on a Sephacryl 5-300 column (Pharmacia LKB Biotechnology Inc.) as described by Mierendorf et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAai. (1983). Fractions containing @-glucosidase activity were pooled and applied to an antibody affinity column prepared using monoclonal antibodies specific to f?-glucosidase (Golumbeski and Dimond, 1986) coupled to CNBr activated Sepharose (Pharma- cia). The enzyme was eluted with 2 M MgClz and dialyzed against 10 r n ~ NaPO. buffer (pH 6). Fractions containing &glucosidase activity were concentrated by a centrifugation using a Centricell device (30,000 M, cut-off, Polyscience) and stored at -20 "C.

Purified @-glucosidase was subjected to SDS-polyacrylamide gel electrophoresis on a 7.5% gel which had been aged 40 h before use (Schatzle et al. 1992). Gel slices containing the 100-kDa mature form of the protein were cut from the gel, inserted into the wells of a 12.5% denaturing polyacrylamide gel, and overlaid with 0.5 pg of V8 protease in 60 pl of Laemmli (1970) sample buffer. Samples were electropho- resed at 15 mA until the dye front reached the separating gel; electrophoresis was then terminated for 30 min to allow protease digestion of @-glucosidase. Following electrophoresis through the sep- arating gel at 30 mA, peptides were blotted to an Immobilon-P membrane (Millipore Co., Bedford, MA) and stained with Coomassie Blue as previously described (Schatzle et al. 1992). The amino acid sequence was determined from blotted p-glucosidase peptides by liquid phase amino acid sequencing (Core Laboratory Facility, LSUMC-New Orleans, LA). Alternatively, the 100-kDa mature form of the enzyme was subjected to SDS-polyacrylamide gel electropho- resis, blotted to Immobilon-P filters as described above, and subjected to N-terminal sequence analysis.

Cloning zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Full-length cDNA Encoding a-Glucosidase-A cDNA library was constructed in phage XZAP using poly(A+) RNA from membrane-bound polysomes (Cardelli et al., 1981). Phage were ab- sorbed onto bacterial strains XL-1 or BB4 and screened using a panel of nine monoclonal antibodies to &glucosidase (Golumbeski and Dimond, 1986) using the protocol recommended by the m~ufacturer (Promega-Bio~h, Madison, WI). The three plasmid clones isolated using this protocol were designated pBG-1, pBG-3, and pBG-16. pBG- 1 was sequenced and was found to code for a partial fragment of the &-glucosidase gene (see Fig. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1). The nucleotide sequence of the pBG- 1 and pBG-16 clones was determined using single-stranded DNA and double-stranded DNA following the sequencing protocols of U. S. Biochemical Corp. AI1 of the sequencing was done using the dide- oxynucleotide chain terminator method and a Sequenase I1 kit (U. S. Biochemical Corp.).

To isolate full-length cDNA encoding &glucosidase, the pBG-1 insert was radiolabeled with 32P and used to screen a different recombinant cDNA library from the one described above constructed in Xgtll with mRNA prepared from cells developing for 4 h (Clon- tech). Probe hybridization and filter washing were performed as previously described (Schatzle et al., 1992). 29 positive plaques were

The abbreviations used are: MES, 4-morpholineethanesulfonic acid; ORF, open reading frame; kb, kilobase.

1470

1

31

61

91

12 1

151

IS1

221

241

271

301

331

361

391

421

451

451

511

541

571

651

631 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

CGTTCCAGMTUCGT-T V P E S R L D

691 - ~ ~ T C T T T ~ A T A ~ ~ M ~ A T A C C M ~ C G C ~ T A G ~ ~ T ~ C C M ~ ~ T ~ T ~ M ~ A C zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

F G D G L S Y T T F N . Y T N L A C S N C X P 1 S G Q S G N Y 1 1

781

811 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBATMAGATT

FIG. 2. The complete DNA sequence of the @-glucosidase cDNA pBG-C; GenBank" accession no. L21014. The numbers to the left designate the amino acid positions from 1 to 821 in the ORF. The seven po~ential N-linked giycosylation sites are denoted by zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAdark zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAclosed zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAcircles while two large predicted hydrophobic stretches are contained within boxes. The two @-glu~sidase peptide sequences determined from sequencing purified glucosidase are u ~ e r ~ i ~ d . The predicted signal sequence cleavage site (amino acid position +24) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAi s represented by a dark triangte and the predicted pro region (+24 to f72) is ~ ~ e r l ~ ~ ~ by dashed lines. The circkd amino acids represent consented residues which probably make up the active site of the enzyme. The arrowheads point to the N-terminal amino acids found in purified mature @- glucosidase.

isolated, and partial DNA sequencing and restriction mapping in&- version 3.5, Macintosh model SE30 computers) designed to determine cated that 20 of these clones were related to each other. One clone protein secondary structure, surface probability, and hydrophobicity. designated as pBG-C was further characterized and found to contain Proteins homology searches were conducted using a computerized three EcoRI fragments 1.6, 0.8, and 0.2 kb in size which were then version of the Lipman-Pearson and Wilbur-Lipman high speed pro- subcloned and subjected to single-stranded dideoxy sequencing. tein library searches (National Biomedical Research Foundation data

quence was analyzed using several computer programs ( ~ a c v e c t o r zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAS o u € h e ~ ~ ~ ~ A ~ ~ s ~ - G e n o m i c DNA was isolated, digested with Protein Analysis-The predicted @-glucosidase amino acid zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAse- base, release number 31.0).

I zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAI 1 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAI zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1 I 1 ' I I I 1 I I I I 1 ' 1 I I 1 I 1 I I I zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAI I I I I I I I zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

100 200 300 400 500 600 700 800

1471

1 I I I I I I I 1 I I I

100 200 300 400 500 600 700 800

FIG. 3. Organization of the predicted &glucosidase precursor. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAThe three predicted domains of the @-glucosidase protein are illustrated in the figure and are designated as the signal sequence, propeptide, and mature protein. Additionally, two large hydrophobic regions (checked boxes) and the predicted active site (dotted box) of the protein are also noted. The amino acid positions from 1 to 821 are also shown below the top schematic of the protein. Potential glycosylation sites are indicated by the lollipop symbol. The hydrophilicity profile of 8-glucosidase was calculated by the Kyte-Doolittle method (1982) based on a window setting of 7. The secondary structure profile of the protein is positioned at the bottom of the diagram. The combined method of secondary structure calculation uses both the Chou-Fasman (1978) and Robson-Garnier (Garnier et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAai., 1978) computer programs.

TABLE I Comparison zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof ~ - g ~ ~ ~ ~ ~ e ~ from different organisms

References for sources of @-glucosidase are as follows: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBALJ. discoideum (this publication); C. t ~ r m o c e ~ ~ m (Grabnitz et at., 1989); I(. marxianus (Raynal et al., 1987); H. anomla (Kohchi and Toh-e, 1985); R. albus (Ohmiya et al., 1990), and S. commune (Moranelli et al., 1986).

mass PI"

kDa

Organism Molecular Percent identity/ Region of similarityb homology'

D. discoideum 89.4 4.77 Bacteria, C. thermocellum 84.1 5.44 29/46 453-821 Yeast, K. marxianus 93.9 5.17 29/42 138-363 Yeast, H. anomla 89.5 4.70 21/36 168-736 Bacteria, Ruminococcus albus 104.3 4.84 24/41 517-695 Fungus, S. commune NDd ND 23 f35 186-486

a Isoelectric point.

e Percent identitylsimila~ty was calculated as described under "Materials and Methods." Region of homology refers to D. d ~ c o ~ e ~ m @-glucosidase amino acid positions, Not determined.

a variety of restriction enzymes, and subjected to Southern blot tometer (LKB 2202 ultrascan, LKB Inc., Gaithersburg, MD). To analysis as described by Schatzle et al. (1992). Blots (Genescreen normalize the levels of @-glucosidase mRNA to ribosomal RNA, the Plus, DuPont NEN) were hybridized in 1XP at 65 "C and washed as blots were stained with methylene blue and quantified as previously described above for the filter lift procedure. BG-1 was nick-translated described (Schatzle et al., 1991). (Life Technologies, Inc.) and used as a probe in both Southern and Northern blot analysis as previously described (Schatzle et al., 1992).

RNA Extractions and Northern Blots-For each developmental or RESULTS AND DISCUSSION

1991). Filters wire exposed to Kodak %kR-5 film at -70 'C, and @: different monoclonal antibodies, all capable of recognizing glucosidase mRNA levels were quantified by a scanning laser densi- mature &-glucosidase on Western blots (Golumbeski and Di-

1472 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBALysosomal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA8-Glucosidase: Sequence zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAand Regulation zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBADevelopment In Suspension Development on Fllterr

2.8 kb 2.7 kb

D.r.,opm.nl 0 1 2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1 4 6 I 14 1 6 1 0 2 0 2 2 24 0 1 2 3 4 I I 14 1818 20 22 0 1 a 2 2 2 4 0

0 2 mRNA

FIG. 4. 8-Glucosidaee mRNA levels are regulated differently in cells developing on filters uersw Ax3 cells developing in suspension. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBATotal RNA was extracted at the indicated time points and subjected to Northern blot analysis using radiolabeled @-glucosidase or D2 DNA inserts as probes (see “Materials and Methods”). The top three panels are the autoradiographs using the @-glucosidase insert as a probe during a developmental time course where cells were in suspension ( f i rst panel) or plated on filters (second panel). The third panel (extreme right) shows the two mRNA @-glucosidase species of 2.7 and 2.8 kb found a t zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAT zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA= 0 and 22/24 h of development. The bottom panels show the autoradioPraDhs of the same two blots shown on the left reurobed with the D2 DNA probe revealing the presence of the 1.8-kb D2 mRNA during the two’time courses.

mond, 1986). The expression library was generated using mRNA isolated from membrane-bound polysomes previously shown to be enriched for mRNAs encoding lysosomal enzymes (Mierendorf et al., 1983). Three positive clones were purified and found to contain inserts (Fig. 1) of 1.1 kb (pBG-l), 1.3 kb (pBG-3), and 1.8 kb (pBG-16). Southern blot analysis indicated that the clones pBG-1 and pBG-16 were related (they cross-hybridized under stringent conditions), and Northern blot analysis indicated that both of these cDNAs recognized a single mRNA species of 2.8 kb (results not shown); the BG-3 clone which hybridized to a 2.0-kb mRNA was not studied further. 2.8 kb is the appropriate size expected for a mRNA encoding a protein of 94 kDa, the molecular mass of unglycosylated @-glucosidase. Southern blot analysis indicated that an antisense oligonucleotide synthesized based on the amino acid sequence of a V8 protease-released peptide from pure @-glucosidase hybridized to both cDNAs (results not shown). Finally, nucleotide sequence analysis of these two clones indicated they were identical where they overlapped, and each contained an ORF coding for an incomplete poly- peptide homologous to @-glucosidase from the yeast Kluyuer- omyces marrianus (Raynal et al., 1987). This suggested that these clones represented incomplete cDNAs encoding D. dis- coideum @-glucosidase. Unfortunately, pBG-16 (containing the largest cDNA insert) represented a fusion clone and contained the extra DNA sequence from nucleotide 1-216 (represented by the dashed line in Fig. 1) which coded for a stretch of amino acids found to be 50% identical to yeast steroyl-CoA desaturase (results not shown).

To isolate full-length cDNAs encoding @-glucosidase, a second Xgtll library prepared using mRNA from cells devel- oping for 4 h was screened using the pBG-1 cDNA insert as a radiolabeled probe. Twenty-nine clones containing inserts ranging in size from 0.2 to 2.6 kb were purified to homogeneity, and partial DNA sequencing and restriction enzyme mapping indicated that 20 of these clones overlapped with pBG-1. The cDNA insert from the largest clone, pBG-C (Fig. l), was sequenced on both strands, and, as described in the next section, this cDNA coded for full-length @-glucosidase.

Southern blot analysis of genomic DNA digested with a variety of restriction enzymes indicated that pBG-C recog- nized only single DNA fragments when used as a probe under moderately stringent hybridization conditions (results not shown). This is consistent with previous genetic data which indicate that D. discoideum @-glucosidase is encoded by a single gene named gluA (Loomis, 1980).

Nucleotide Sequence and Deduced Amino Acid Sequence zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof the @-Glucosidase cDNA, BG-C-The 2594-nucleotide pBG-C cDNA insert was sequenced on both strands as described under “Materials and Methods.” A single long ORF of 2463 nucleotides coding for 821 amino acids was detected extending from nucleotide 68 to nucleotide 2594. The ORF begins with an ATG (nucleotides 67-69) and ends with a TAA stop codon suggesting it coded for a full-length protein. This first ATG most likely represents the initiation codon because it is pre- ceded by a very A/T-rich region containing stop codons in all three reading frames; translation initiation codons are usually preceded by a string of As in D. discoideum.

Conceptual translation of this long ORF yielded a predicted protein of 89-kDa molecular mass and an isoelectric point (PI) of 4.8. This is close to the molecular mass of the @- glucosidase primary translation product of 94 kDa (Cardelli et al., 1987) and the experimentally determined PI of the protein (results not shown). The predicted amino acid se- quence also matched the sequence of the two V8-derived @- glucosidase peptides (represented by the single underlines in Fig. 2) indicating the pBG-C clone codes for authentic @- glucosidase. Finally, seven possible N-glycosylation sites were located throughout the length of the polypeptide (Figs. 2 and 3) which agrees well with the experimentally determined number of six N-linked oligosaccharide side chains per pre- cursor (Cardelli et al., 1986).

The predicted @-glucosidase amino acid sequence was used to search the NBRF protein sequence data base to identify homologous proteins. The search revealed that the polypep- tide contained long stretches of amino acid sequence that were significantly homologous to @-glucosidase amino acid sequences identified from both a yeast species, K. marxianus (Raynal et al., 1987), and a bacterial species, Clostridium thermocellum (Grabnitz et al., 1989) (Table I). Interestingly, these homologous sequences did not overlap in position be- tween the yeast and bacterial proteins. For instance, the D. discoideum region homologous to the yeast @-glucosidase (29% amino acid sequence identity and 46% similarity) was located in the N-terminal portion of the D. discoideum protein and spanned amino acid positions 138-363. In contrast, the region homologous to Clostridium @-glucosidase (29% amino acid identity and 46% similarity) was found in the C-terminal portion of the slime mold protein (amino acid positions 453- 821). The D. discoideum @-glucosidase amino acid sequence was also homologous to a lesser extent to @-glucosidases from other species (Table I). However, no significant sequence

Lysosomal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAP-Glucosidase: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAhomology was shared between the D. discoideum @-glucosidase and human glucocerebrosidase (results not shown). Interest- ingly, the predicted molecular masses and isoelectric points of the @-glucosidase enzymes from the six listed organisms (Table I) were very similar (ranging from 89 to 104 kDa in molecular mass and 4.7 to 5.4 in isoelectric point, respec- tively).

Predicted Structural Features zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof the @-Glucosidase Polypep- tide-Fig. 3 indicates a schematic diagram of the predicted domains of the D. discoideum @-glucosidase polypeptide along with a computer assisted analysis indicating hydrophobic regions and regions predicted to have secondary structure. The most hydrophobic region was found at the N terminus (amino acid positions 4-21) and contained features indicating that it may function as a signal sequence (Fig. 2). For instance, positively charged lysines were found at amino acid positions zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

A 1 . o zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0-0 Suspension zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA0 . 8

0 . 6

0 . 4

0 . 2

0 . 0 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

B Time of Development (Hours) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

CHJ Suspension 0 . 8 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

e 0

0 .

0 . 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

Time of Development (Hours)

MORPHOLOGY A *- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA9 STAGE Preagg---Aggregation---Slug--Culmination--Completion

FIG. 5. Graphical representation of laser densitometric scans of the autoradiographs in Fig. 4. Panels A and B illustrate the relative levels of P-glucosidase (in suspension, open circles, or on filters, closed circles) and D2 mRNA (in suspension, open squares, or on filters, closed squares), respectively, as determined by laser densi- tometric analysis of autoradiographs and normalized to the levels of ribosomal RNA in each lane of the probed Northern blots (Schatzle et al., 1993).

Sequence and Regulation 1473

2 and 5 followed by a stretch of 19 uncharged amino acids (11 which were hydrophobic). Based on the rules proposed by von Heinje (1984) we predict that cleavage of the signal sequence occurs on the carboxyl side of Gly24. A very hydrophylic stretch of 47 amino acids extending from amino acid positions 25-71 immediately followed the putative signal sequence (dot- ted underline in Fig. 2; striped box in Fig. 3); 15 charged amino acids and 14 polar amino acids were distributed throughout this span. The 105-kDa @-glucosidase precursor is cleaved intracellularly first in a Golgi/endosomal compartment to a 103-kDa intermediate form and then in lysosomes to a 100- kDa mature form. Since the molecular mass of this hydro- phylic stretch of amino acids is approximately 5 kDa, we reasoned that it might be a “pro” region subject to proteolytic removal. To directly test this hypothesis, we purified the mature 100-kDa @-glucosidase protein as described under “Materials and Methods” and determined the N-terminal amino acid sequence for 8 residues. Two N termini were present in the purified preparation and corresponded to Ile7’ and Ser74. Since the processing of the 103-kDa intermediate form of @-glucosidase is catalyzed by a cysteine proteinase(s), we propose that the actual cleavage site generating the mature subunit is at the L y ~ ~ ~ L y s ‘ j ~ pair and that an N-terminal peptidase generates the two different mature N termini. How- ever, we cannot rule out the possibility that these N termini were generated artifactually during purification of the protein.

The processing of the 105-kDa precursor to the 103-kDa intermediate form is catalyzed by a proteinase having prop- erties similar to yeast kex2 and the Lys4’Ar$’ pair represents a possible cleavage site for this processing event (Richardson et al., 1988). Interestingly, The D. discoideum a-mannosidase lysosomal enzyme also contains a short N-terminal hydro- phylic pro region contiguous with the signal sequence that has been demonstrated by direct amino acid sequence of the mature 60-kDa a-mannosidase subunit to be removed in vivo. This pro region also contains an amino acid pair that should be recognized by a kex2 proteinase (Fuller et al., 1989). Inter- estingly, the size, location, and charge properties of D. discoi- deum a-mannosidase and @-glucosidase pro regions are simi- lar to both yeast and plant hydrolase pro regions that have been found to be necessary and sufficient for localization to lysosomes (Klionsky and Emr, 1990; Matsuoka and Naka- mura, 1991). Although it is reasonable to propose that these pro regions play a role in targeting of hydrolases to lysosomes in Dictyostelium, it should be noted that the targeting infor- mation for the slime mold @-hexosaminidase enzyme does not reside in a pro region (LaCoste et al., 1992; Graham et al., 1988) (in fact @-hexosaminidase is not cleaved at all during transit to lysosomes).

In addition to the putative signal sequence, the @-glucosi- dase polypeptide contained two other hydrophobic stretches greater than 14 amino acids in length (boxed amino acids in Fig. 2, and hatched boxes in Fig. 3). The first region may form a @-sheet structure that extends from amino acid position 216 to amino acid position 235 (with a hydrophobic index greater than 2.0). The second region extends from amino acid position 385-416 and also contains subregions with hydrophobic in- dices greater than 2.0. Unlike the first region, the region may assume an a-helical structure. These two regions may be responsible in part for the association observed in vivo be- tween the precursor of @-glucosidase and intracellular mem- branes; a-mannosidase also contains hydrophobic stretches that may function to anchor the precursor to membranes. In theory, both of these regions are long enough to span the lipid bilayer, however, neither a-mannosidase nor @-glucosidase

1474 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBALysosomal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA/3-Glucosidase: Sequence zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAand Regulation zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBANC4 A x 4 A x 3 G a 2

Development Hours Of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA0 2 4 1 0 2 4 7 1 5 0 2 6 1 2 0 2 6 1 2

CsA zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA- ” + ”++ ” “ + ””

FIG. 6. &Glucosidase mRNA levels decrease in cells that become aggregation competent. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBATotal RNA was extracted at the time points in development indicated below each panel from the D. discoideurn strains, NC4, Ax4, Ax3, and Gn2 and subjected to Northern blot analysis using radiolabeled 8-glucosidase DNA as a probe. The aggregation competence state of the cells is shown below each time point as either CsA + (aggregating) or CsA - (non-aggregating).

Cells Grown:

(F

Axenically Bacterial Food Source I l l , , , , I j Hours of

Development 0 2 4 7 1 5 2 2 2 6 0 2 4 7 1 5 1 7 2 2 2 6

FIG. 7. The pattern of &glucosidase expression is the same during development regardless of how cells were grown prior to the initiation of development. Ax3 cells which were grown axenically or on bacterial-seeded plates were harvested and plated on filters to initiate development. Total RNA was extracted at the indicated time points and subjected to Northern blot analysis using radiolabeled zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA8- glucosidase DNA as a probe. The two panels are the autoradiographs from one set of these experiments. The left panel shows the levels of p- glucosidase mRNA in cells previously grown axenically while the right panel indicates the mRNA levels in cells grown on bacteria.

are likely to be integral membrane proteins (Cardelli, 1993) suggesting these hydrophobic regions probably do not cross lipid bilayers.

The active site of @-glucosidase from Aspergillus wentii has been identified (Bause and Legler, 1980) and shown to contain an invariant Asp separated from a conserved Glu 10 amino acids upstream. Fig. 2 indicates the amino acid residues which might form the active site in D. discoideum @-glucosidase. Trp and Gly amino acid residues also appear to be conserved in addition to the Asp and Glu residues for @-glucosidase from a variety of organisms including D. discoideum (Fig. 2), Hun- senula anomala (Kohchi and Toh-e, 1985), Schizophyllum commune (Moranelli et al., 1986), K. marxianus (Raynal et al., 1987) and C. thermocellum (Grabnitz et al., 1989).

Developmental Regulation zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof the @-Glucosidase Gene-Pre- vious studies indicated that the relative levels of translatable @-glucosidase mRNA and enzyme activity are high in growing cells and increase during early development, followed by a decrease to negligible levels during aggregation and slug mi- gration, and a return to high levels during the final stages of development (Coston and Loomis, 1969; Golumbeski and Dimond, 1987). To more accurately determine the changes in @-glucosidase gene expression during development, RNA was extracted from growing cells and from cells a t various times during development, and subjected to Northern blot analysis. The “P-radiolabeled BG-1 insert was used as a probe. Our laboratory Ax3 wild-type cells were allowed to develop under two different conditions: 1) on filters under which they com- plete the entire developmental cycle and form fruiting bodies and 2) in a buffered salt solution where normally cells reach only the aggregation competent stage. Fig. 5 indicates that high levels of @-glucosidase mRNA were observed in growing cells and in cells during early development on filters (T = 0- 4 h). The levels of mRNA dramatically decreased at the beginning of the aggregation stage (T = 6-8 h) of development (the lawn of cells had begun to “ripple,” an indication of

beginning aggregation). Cells completed aggregation and formed tight mounds by T = 12 h. Steady state levels of @- glucosidase mRNA remained very low until the culmination stage (7‘ = 16-18 h) of development a t which time a greater than 50-fold increase was observed Fig. 5 is a laser densito- metric scan of the autoradiographs of Fig. 4 to quantify the data. These data correspond well with published reports (Gol- umbeski and Dimond, 1987; Loomis, 1975) on the regulation of levels of @-glucosidase enzyme activity, although the in- crease in mRNA during early development reported here was not as dramatic as previously reported. Interestingly, the @- glucosidase mRNA that reappeared late in development was 100-150 nucleotides larger than the @-glucosidase mRNA present early in development (see far right panel of Fig. 4 which indicates mRNA from three late time points bracketed by RNA from vegetative cells). The reason for the differences in size of the mRNAs is presently unknown but may include alternative post-transcriptional processing and/or use of al- ternative promoters. In contrast to the results described above for cells developing on filters, steady state levels of @-gluco- sidase mRNA dropped 50% in the 1st h and then decreased slowly during the next 20 h in Ax3 cells suspended in a buffered salt solution (Figs. 4 and 5 ) ; only a slight increase in the level and no change in the size of the mRNA was observed after 20-24 h of starvation.

The Northern blots described above were stripped of DNA and reprobed with a second radiolabeled cDNA, D2, whose encoding gene is transcriptionally induced in cells actively sending and receiving CAMP pulses, a developmentally in- duced signaling pathway required for chemotaxis and aggre- gation. Levels of D2 mRNA rapidly increased at the beginning of aggregation (T = 4-6 h) in cells developing on filters (Figs. 4 and 5). In contrast, our Ax3 wild-type cells suspended in a salt solution contained significantly lower levels of accumu- lated D2 mRNA by 6-8 h, suggesting these cells did not completely become aggregation competent. This was con-

~ y s o s o ~ u Z zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA~ - G l u c o s ~ ~ e : Sequence and ~ e g u ~ ~ ~ ~ n zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1475 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAfirmed by an experiment which indicated that Ax3 cells in suspension (in contrast to cells on filters) accumulated only low levels of the cell surface glycoprotein zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAgp80 (also termed contact sites A or CsA) which is required for cell adhesion during aggregation and accumulates at that time (results not shown). Also, cells recovered from suspension did not form polar end to end contacts (typical of aggregation competent cells) nor did they form aggregation streams when plated on plastic dishes (results not shown). These data suggest that the decline in steady state levels of @-glucosidase mRNA depended on cells becoming ag~gation-competent, and that our laboratory Ax3 strain did not reach this stage of devel- opment when starved in a buffered salt solution. To further test this, two other wild-type strains, Ax4 and NC4, shown by others to be capable of becoming aggregation competent in suspension (Loomis, 1982), were placed under the devel- opmental conditions described above. Northern blot analysis (Fig. 6) of RNA isolated from these strains during develop- ment in suspension indicated that at the time (T zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA= 8-10 h) cells became aggregation competent (Le. CsA positive), 0- glucosidase mRNA levels rapidly dropped. Furthermore, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA8- glucosidase mRNA levels did not decrease in a mutant strain deleted of the Ga2 gene (Kumagai et ai., 1991) when cells were starved on filters (Fig. 6) or in suspension (results not shown). Ga2 is a subunit of a heterotrimeric zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAG protein com- plex that is functionally coupled to the cyclic AMP receptor (cAR1) and is necessary for cAMP signal transduction re- quired for aggregation. Therefore, the decrease in levels of p- glucosidase mRNA depended on cells acquiring the ability to respond to cAMP signals and becoming aggregation compe- tent regardless of whether this event occurred in suspension or on filters (i.e. development on filters is not an absolute requirement to affect a decrease in the level of @-glucosidase mRNA). Moreover, the increase in concentration of a larger size 8-glucosidase mRNA occurring at approximately 16-18 h of development may depend on the ability of cells to enter the culmination stage which only occurred in cells developing on filters.

The developmentally induced decrease in the levels of cer- tain zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAD. discoideum mRNAs depends on the prior growth conditions of the cells. For instance, in cells grown axenically, the steady state levels of mRNAs coding for ribosomal pro- teins remain constant until developing cells reach the aggre- gation stage at which time mRNA levels rapidly and coordi- nately decrease (Steel and Jacobson, 1987). In contrast, in cells grown in association with bacterial as a food source, levels of ribosomal protein mRNAs rapidly decrease upon the initiation of development (Singleton et al., 1989). As indicated in the Northern blot of Fig. 7, @-glucosidase mRNA levels decreased at 4-7 h of development (beginning of aggregation) and increased again at 22 h of development (culmination stage) regardless of whether cells were grown axenically or in association with bacteria. Additionally, regardless of the con- ditions used to grow cells both the 2.7- and 2.8-kb @-glucosi- dase mRNAs were observed at the appropriate times during development. This suggests that prior growth conditions do not influence either the timing of the decrease and subsequent increase in levels of both forms of the @-glucosidase mRNA and that the mechanisms regulating the decrease in levels of @-glucosidase mRNA are different than the mechanisms reg- ulating expression of the ribosomal protein.

CONCLUSION

We report here the cloning and DNA sequence analysis of the cDNA encoding the full- len~h precursor polypeptide to

lysosomal @-glucosidase from D. discoideum. The predicted protein contained regions homologous to both fungi and bac- terial @-glucosidases and also contained a potential N-termi- nal signal sequence followed by a short very hydrophylic “pro” region similar in location and size to the pro-I domain of D. discoideum a-mannosidase. Short N-terminal localized hydro- phylic pro regions function to target hydrolases to lysosomes in plants and yeast cells. The @-glucosidase precursor also contained hydrophobic regions that might facilitate associa- tion of the protein with intracellular membranes. The expres- sion of the gene encoding lysosomal @-glucosidase was under a relatively complex mode of regulation during develop men^ the relatively high steady state levels of mRNA in growing and early developing cells decrease by the aggregation stage of development to undetectable levels. This reduction in mRNA levels depended on cells acquiring the ability to aggre- gate. This was followed by a significant increase during the final stages of development in the levels of a @-glucosidase mRNA larger in size than that observed in early development.

Acknowledgments-We thank Dr. Richard Firtel for the D2 cDNA and the Ga2 mutant cell lines. We also thank members of the Cardelli laboratory for critical reading of the manuscript, and we thank Karl Franek and Dr. Mike Wolcott for aiding in the purification of 8- glucosidase. We recognize the support of the LSUMC Centers for Excellence in Cancer Research, and for Excellence in Arthritis and Rheumatology.

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