Proteolytic processing of the pro0 chain of 8-hexosaminidase occurs at basic residues contained within an exposed disulfide loop structure

CARMELINA SAGHERIAN, STEPHEN POROSZLAY, GEORGE VAVOUGIOS, AND DON MAHURAN'

Research Institute, Hospital for Sick Children, 555 University Ave, Toronto, ON M5G 1x8, Canada

and

Department of Clinical Biochemistry, University of Toronto, Toronto, ON M5S IA8, Canada

Received April 10, 1993

SAGHERIAN, C., POROSZLAY, S., VAVOUGIOS, G., and MAHURAN, D. 1993. Proteolytic processing of the proP chain of P-hexosaminidase occurs at basic residues contained within an exposed disulfide loop structure. Biochem. Cell Biol. 71: 340-347.

Lysosomal P-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes, Hex A (aP) and Hex B (PP) The a and /3 subunits are encoded by the HEXA and HEXB genes, respectively. Extensive homology in both the gene structures and deduced primary sequences demonstrate their common evolutionary origin. While undergoing similar proteolytic modifications in the lysosome, the pro0 polypeptide is additionally cleaved internally to produce the mature 24-30 kilodalton Pb and Pa chains. Previous data have suggested that this processing event occurs somewhere between residues Ser311 and Lys315. In this report we demonstrate that this area is located in a hydrophilic disulfide-loop structure (between Cys309 and Cys360). The cleavage event is prevented by the deletion through in vitro mutagenesis of the Arg312-Gln-Asn-Lys tetrapeptide or by its substitution with the aligned a residues (Gly-Ser-Glu-Pro). Reintroduction of either Arg312 or Lys315 reinstates the processing. Furthermore, we show that this area is not involved in lysosomal targeting of pro-Hex B, or in the increased stability or the variation in substrate specificity of the 0 as compared with the a subunit. Our data suggest the presence of a novel lysosomal endoprotease. Like other endoproteases it is specific for basic amino acids; however, it cleaves on the amino-terminal side rather than the conventional carboxy-terminal side of such residues and then only if they are fully exposed to the lysosomal environment. These data are consistent with the idea that the partially degraded enzyme resulting from exposure to the harsh environment of the lysosome is the resistant, but still functional product of the protein's adaptation to its lysosomal role. However, the residues that are removed appear to have been necessary for the initial folding and disulfide bond formation of the propolypeptides in the endoplasmic reticulum.

Key words: lysosomal, glycosidase, endoprotease, maturation, transport.

SAGHERIAN, C., POROSZLAY, S., VAVOUGIOS, G., et MAHURAN, D. 1993. Proteolytic processing of the prop chain of P-hexosaminidase occurs at basic residues contained within an exposed disulfide loop structure. Biochem. Cell Biol. 71 : 340-347.

I1 existe deux isoenzymes principales de la 0-hexosaminidase (EC 3.2.1.52) dans les lysosomes : Hex A (ap) et Hex B (OD). Les sous-unites a et P sont codees respectivement par les genes HEXA et HEXB. L'origine commune de ces deux genes au cours de I'evolution est demontree par la t r b grande homologie de leurs structures et de leurs sequences primaires deduites. En plus des modifications proteolytiques similaires dont sont I'objet les produits de ces genes dans les lyso- somes, le polypeptFe pro0 subit une coupure intramoltculaire, ce qui produit les chaines 6, et Pb matures de 24-30 kilodaltons. A partir des donnkes anterieures, il a ete suggere que ce clivage se produisait quelque part entre les rksidus Ser311 et Lys315. Dans cet article, nous demontrons que cette region est situee dans la structure hydrophile en boucle cr&e par un pont disulfure (entre Cys309 et Cys360). Le clivage est impossible a la suite de la deletion du tetrapeptide Arg312-Gln-Asn-Lys par mutagenese in vitro ou la suite de sa substitution par des groupements a alignes (Gly-Ser-Glu-Pro). La reintroduction de l'Arg312 ou de la Lys315 rend de nouveau possible le clivage. De plus, nous montrons que la region de clivage n'est pas impliquee dans le ciblage de la proHex B vers les lysosomes ou pour la stabilite plus grande ou la specificit6 de substrat diffkrente de la sous-unite /3 par rapport A la sous-unite a. Nos resultats suggerent qu'il y a une nouvelle endoprotease dans les lysosomes. Comme d'autres endoproteases, elle est specifique d'acides amines basiques; cependant, alors que c'est la liaison peptidique du cBte carboxyterminal de ces acides amines qui est habituellement hydrolysee, cette nouvelle endoprotiase coupe plutBt celle du c6te aminoterminal et seulement si les substrats sont completement a l'interieur des lysosomes. Ces resultats concordent avec le concept que I'enzyme partiellement degradee, resultant de l'exposition au milieu rigoureux des lysosomes, est le produit resistant mais encore fonctionnel de l'adaptation de la proteine a son r61e dans les lysosomes. Cependant, les residus enleves semblent neces- saires pour le repliement initial et la formation du pont disulfure des propolypeptides dans le reticulum endoplasmique.

Mots elks : lysosome, glycosidase, endoprotease, maturation, transport. [Traduit par la redaction]

ABBREVIATIONS: Hex, P-hexosaminidase; 4-MUG, 4-methylumbelliferone-PN-acetylglucosamine; CMUGS, 4-methylumbelli- ferone-P-N-acetylglucosamine-6-sulfate; GalNAc, N-acetylgalactosamine; kDa, kilodalton(s); HPLC, high performance liquid chroma- tography; TFA, trifluoroacetic acid; kb, kilobase(s); bp, base pair(s); a-MEM, a-minimal essential media; CAT, chloramphenicol acetyltransferase; IgG, immunoglobulin G; HSA, human serum albumin; M,, relative mass.

' ~ u t h o r to whom all correspondence should be sent at the following address: Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, ON MSG 2x8, Canada. Printed in Canada / Imprim6 au Canada

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SAGHERIAN ET AL. 341

Introduction Hex is a lysosomal glycosidase that removes terminal

0-linked N-acetylglucosamine and N-acetylgalactosamine residues from a variety of natural and synthetic substrates. In normal human tissues, it exists in two major isozyme forms, Hex A (a@) and Hex B (00). The a and 0 subunits are encoded by two distinct, but evolutionarily related genes, HEXA and HEXB, respectively. Thus, their primary struc- tures show an overall 60% homology. While dimer forma- tion is required for either subunit to become active (Mahuran and Lowden 1980), both the a and 0 subunits contain active sites that are able to hydrolyze many of the same neutral artificial (e.g., 4-MUG) and natural substrates. However, only the catalytic site in the a-subunit can hydrolyze nega- tively charged substrates such as 0-linked glucosamine 6-sulfate containing glycosaminoglycans (Kresse et al. 1981) and artificial substrates (e.g., 4-MUGS) (Bayleran et al. 1984), and most importantly GMz ganglioside (GalNAcP- (1 -4)-(NANAa(2-3)-) -GalP(l-4)-Glc-ceramide). Although any combination of subunits can produce an active enzyme, the presence of the 0 subunit greatly increases the stability of the resulting dimer (reviewed in Mahuran 1991).

The structure of the a and subunits found in Hex isozymes isolated from tissues differs from that deduced from the cDNAs. Both subunits undergo posttranslational modifications in the endoplasmic reticulum and the Golgi apparatus characteristic of secretory proteins, e.g., signal peptide cleavage, Asn-linked glycosylation, formation of intrachain disulfide bonds, and subunit association. In addi- tion, lysosomal proteins must obtain a mannosed-phosphate recognition marker in the salvage compartment and cis Golgi, to be sorted from secretory proteins in the trans Golgi network (reviewed in Kornfeld 1990).

As a characteristic of many lysosomal enzymes, the major structural modifications converting the dimeric pro-Hex isozymes to their mature forms occur in the lysosome through proteolytic and glycosidic processing. The amino termini of the two mature a and three mature 0 chains (some of their carboxyl termini remain speculative and are indi- cated by " - " below) and several stable intermediates have been determined by amino acid sequencing of the (i) purified components of the Hex isozymes isolated from placenta (Mahuran et al. 1988; Hubbes et al. 1989; Mahuran 1990) o r (ii) biosynthetically radiolabelled components immunoprecipitated from cultured fibroblasts (Little et al. 1988; Quon et al. 1989; Sonderfeld-Fresko and Proia 1989). The 67-kDa proa polypeptide is processed to a -7-kDa chain (a,, residues 23 to - 74 in the preproa sequence) and a glycosylated 56-kDa mature a polypeptide (a,, residues 89 to 529). The 63-kDa pro0 polypeptide is also cleaved at a similar position to produce a 7- to 15-kDa glycosylated 0, chain (residues 50 to - 107 in the prepro0 sequence). In addi- tion, there is an internal cleavage unique to the pro0 chain t o produce the mature glycosylated polypeptides Pb (24-26 kDa, residues 122 to - 31 1) and Pa (28-30 kDa, residues 315 to 556). It has been suggested that this unique 0-cleavage removes a tetrapeptide, Arg3 12-Gln-Asn-Lys3 15, and a tripeptide, Arg312-Gln-Asn, from the pro0 chain of Hex A and Hex B, respectively (Mahuran et al. 1988) (Fig. 1). However, more recent data indicates that the removal of Lys315 as the amino terminus of the 0, chain in Hex A occurs at a later stage of maturation (Mahuran 1990). Thus, the initial generation of the Pb and 0, chains maximally involves the removal of the tripeptide.

The biological reason for the proteolytic processing in the lysosome is not clear. These events are not required for enzymatic activity since the precursor forms are catalytically active (Hasilik et al. 1982; Mahuran 1990). However, in yeast a precursor-specific domain in proteinase A has been dem- onstrated to be involved in targeting the protein to the vacuole (Klionsky et al. 1988). Thus, a possible initial role for these prosequences in targeting Hex to the lysosome, i.e., as a signal for oligosaccharide phosphorylation, requires examination. To probe the function of this protein domain and determine the mechanism of proteolysis that cleaves the prop, but not the proa (despite the close homology sur- rounding the predicted cleavage site (Fig. I)), we have employed in vitro mutagenesis and expression in COS-1 cells.

Materials and methods Isolation of tryptic peptides joined by a disulfide bond

Purified placental Hex B (10 mg) was trypsinized as previously described (Korneluk et al. 1986), except that the denatured protein was not reduced prior to its digestion. HPLC separation of the resulting peptides was also carried out as previously described (Korneluk et al. 1986). Final purification of the peak of interest was obtained by reisolation of the peak from a shallow 26-29% solvent B (isopropanol-acetonitrile, 1:2 v/v containing 0.065% TFA) gradient elution of the reverse-phase (C-18) column (sol- vent A, 0.1% TFA in dH20), followed by another reisolation using an isocratic elution at 26% solvent B. Partial amino acid sequencing (five turns) was carried out as previously reported (Korneluk et al. 1986).

Oligonucleotide-directed in vitro mutagenesis General cloning procedures were as described by Maniatis

(Sambrook et al. 1989). A 1.3-kb EcoRV/PstI cDNA fragment of the @ subunit from pHexB43, containing the cDNA sequence encoding the @ subunits of @-Hex (Korneluk et al. 1986), was subcloned into pBS + (Stratagene) for constructs i-iv and pBS - for construct v (see Table 1). Single-stranded DNA was prepared by superinfection with M13 helper phage. Oligonucleotides, cor- responding to the sense strand for constructs i-iv and the antisense strand for construct v, were synthesized to introduce various modifications into the putative tripeptide "clip site" and the adjoin- ing Lys315 residue, i.e., the tetrapeptide Arg312-Gln-Asn-Lys (R,,,QNK) of the pro@ chain of Hex B: (i) 5'-CTGACTCCATG- TTACAGTTTGGACTCTTTTGGACCT-3 ' , a 36-mer, for the deletion of the four residues to produce construct AR,,,QNK; (ii) 5 ' -AAAGACCTCCTGACTCCATGTTACAGTGGAT- CAGAGCCGTTGGACTCTTTTGGACCTATAAACCCT-3 ' , a 66-mer, to substitute the four @ residues with the residues found at their aligned positions (Fig. 1, a-Gly280-Ser-Glu-Pro) in the deduced sequence of the human cr-chain of Hex A producing construct a (GSEP) (i.e., P-Gly3 12-Ser-Glu-Pro); (iii) 5 ' -ATG'I?T- ACAGTAGATCAGAGCCGTTGGA-3 ' , a 27-mer, to resubstitute the @-Arg312 residue for the a-derived Gly residue in the "a- sequence substitution" cDNA construct, producing construct Arg-a (i.e., RSEP); (iv) GGATCAGAGAAGTTGGACTCTTTTGGAC- CT-3', a 30-mer, to resubstitute the 0-Lys315 residue for the a-derived Pro residue in the a sequence substitution cDNA con- struct, producing construct a-Lys (i.e., GSEK); and (v) 5'- CTTGTTTTGTTGACTGTAAC-3 ' , a 20-mer, to substitute Gln for Arg312 in the wild-type 0 sequence, producing construct Q,,,QNK (i.e., Gln312-Gln-Asn-Lys). The in vitro mutagenesis was performed as previously reported (Brown et al. 1989). The mutations were verified by sequencing approximately 900 bp (including the mutation site) of the fragment using the sang& dideoxy-chain termination method. To undergo the expression studies, the mutant fragments were subcloned back into pHexB43, a pCD-derived plasmid with the SV40 early region promoter and a modified SV40 late region intron necessary for expression in mammalian cells (Okayama and Berg 1982).

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342 BIOCHEM. CELL BIOL. VOL. 71, 1993

a (HI PGLLTP

&I*= p (HI KDLLTP

. .... . .... p (M) KNLLTP

p (DDIPELVAT

F I G . 1. Related Hex sequences aligned with the internal site of hydrolysis of the proP chain are shown in an oval; note the total lack of sequence homology in this area of the chain of human Hex (P (H)) and corresponding areas in the a subunit of human Hex (a (H)), the fl subunit from mouse Hex (0 (M)), and the p subunit from Dictyosteliurn discoideurn (0 (DD). The fourth oligosaccharide attachment site in the human pro0 chain, which is the primary site of phosphorylation necessary for lysosomal targeting (Sonderfeld- Fresko and Proia 1989), is boxed along with homologous sites in the human a and mouse subunits. The disulfide loop structure identified in this report is boxed; note the conservation of Cys residues and the size of the loop in all sources of Hex subunits. Partial amino acid sequences, five turns of the sequenator, of a single peak from a reverse-phase HPLC column (Fig. 2) are underlined. The two sets of five amino acids constituting the partial sequences of two tryptic peptides were present in equimolar ratios.

Internal s-s Cleavage

TABLE 1. Specific activity and heat stability of the human &Hex B synthesized by transfected COS cells

Intracellular Hex B Activity secreted (nmol/(h.pg)) Hex B activity TI /2

(qo of total), fib/@, at Free Bound bound cleavage 60°C

Constructsa (COS) (human) (human) (Fig. 3) (min)

(i) AR3,,QNK 0.5k0.1 4.920.7 12.1k0.7 - 4.6 k 0.2 (ii) a (G3],SEP) 0.6k 0.2 4.8k 0.2 13.6k 0.6 - 15.8k0.8 (iii) Arg312-a 0.4+0.1 5.4k0.4 13.9k3.0 + + 14.3k0.7 (iv) a-L~s315 0.3kO.l 7.0k0.9 15.6k0.9 + + 15.4k6.0 (v) Q3,,QNK 0.5 k 0.1 4.8 k 0.5 N D ~ + + + + ND

YS GSE PSGTFGPVNPS EF'MSTFFLEVSSVFPDFYLHLGGDEVDFT

YS RQN KLDSFGPLNPT '[[E!liK . . . . . . . . . . . . . . . . . . YNQKT KTQVFGWDP T AFFNTFFKEISVDPTVNTTHLGGDEVEFQ

. . .. . .... . . . * .

PD YAA NVNNI-PLDISNPATFTFIQNLFTEIAPLFIDNYFHTGGDELVTG

COS(+ )C 0.4 3.4 29.3 + + + + 17.1k3.5 COS( - )d 0.4 0.06 Hex Be 21.4

"COS cell extracts were analyzed and the results were averaged from three transfection experiments. The Hex B synthesized were from four mutant pHexB43 cDNA constructs encoding a fl subunit containing (i) a deletion of the residues spanning the cleavage site, i.e., construct AR312QNK; (ii) a substitution of the four B residues with the aligned residues from the human a subunit, i.e., construct a (GSEP) (Fig. 1); (iii) the replacement of one basic B residue for the amino-terminal o residue in the substitution mutation, i.e., construct Arg-Ser-Glu-Pro (Arg-a); (iv) the replacement of the other basic B residue for the carboxyl- terminal a residue in the substitution mutation, i.e., construct Gly-Ser-Glu-Lys (a-Lys); and (v) replacement of Arg312 with Gln in the normal fl sequence, i.e., construct Q312QNK.

b ~ o t determined. 'Extract from COS cells transfected with wild-type ((+), control) pHexB43, containing the sequence

RII~QNK. d~xtract from untransfected COS cells ((-), control). ePurified from human placenta (Mahuran and Lowden 1980).

C

. . C . . C -

Cell culture Solid state irnrnunoprecipitation assay for Hex COS-1 monkey kidney cells (American Culture Collection) were This procedure utilizes a human-specific immunoprecipitation

maintained in a-MEM (Flow Laboratories) supplemented with assay system to eliminate the contribution of the endogenous COS streptomycin and penicillin (100 mg/mL) and 10% nondialyzed enzyme activity (Brown et al. 1989; Neote et al. 1990). Briefly, COS fetal bovine serum at 37OC in 5% CO,. lysate or cell culture medium was rotated at 4°C overnight with

25 uL of a ~rote in A - Se~harose bead sus~ension (Pharmacia)

WKsso -

iE3K4 . . +

WA339 + . . WL314 -

DNA transfections COS-1 cells were transfected using 10 pg of pHexB43 containing

either the wild-type insert or inserts encoding the various clip-site mutations. All the cDNA constructs were cotransfected with 10 pg of pBLCAT2, a pUC18-derived plasmid containing the coding region of the bacterial CAT gene, used to monitor transfection efficiency from one cell culture dish to another (Neumann et al. 1987). Transient expression was carried out for 48 h and cells were harvested as described (Neote et al. 1990).

and 10 &'of a polyclonal rabbit anti-human Hex B I ~ G ( - 7 mg/mL). After centrifugation the supernatant was removed and analyzed for COS Hex activity (to ensure the total precipitation of the human-derived protein), while the bound immune complex was washed three times with phosphate-buffered saline and assayed directly for human Hex activity using the common artificial sub- strate 4-MUG or the a-specific artificial substrate 4-MUGS.

Western blot analysis Western blots were performed as previously described (Brown

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SAGHERIAN ET AL. 343

et al. 1989; Neote et al. 1990). Lysates were analyzed using equal amounts of human Hex activity (240 nmol/h).

Stability studies The amount of transfected COS lysate used was predetermined

by the solid state immunoprecipitation assay described above. The appropriate amoung of lysate (Brown et al. 1989; Neote et al. 1990), which was dependent on transfection efficiency, was then incubated at 60°C in 0.3% HSA and citric acid - phosphate buffer (pH 4.1). Aliquots (75 pL) were removed at 0, 5, 10, 15, and 20 min, and assayed using the solid state immunoprecipitation assay for Hex to determine the residual amount of human Hex B activity at each time point (Brown et al. 1989; Neote et al. 1990). The log(% residual activity) was plotted against incubation time. From these data a best-fit line was generated by the least-squares method, and the time t,, , (in minutes), at which 50% of the original activity remained, was calculated.

Results Characterization of two tryptic peptides joined by a

disulfide bond Since the three mature 0 chains are held together in the

0 subunit by disulfide bonds (Mahuran and Lowden 1980; Hubbes et al. 1989) and the mature Pb chain contains only two Cys residues (Cys91 and Cys309). at least one of these Cys must be involved in an interchain disulfide bond. Cys309 is near the carboxyl terminus of the Pb chain, within three residues of the proposed Pb-& cleavage site. We fortui- tously obtained information as to its status within the mature 0 subunit. Major tryptic peptides, generated from non- reduced Hex B, were separated by reverse phase HPLC. After a comparison was made to the HPLC separation pattern of fully reduced, trypsinized Hex B (Korneluk et al. 1986; Mahuran et al. 1988), there was only one major new peak observed, 0-14 eluting at 41 min (data not shown). It was further purified to a symmetrical peak by a shallow 26-29% solvent B gradient elution (Fig. 2A, 0-14-5), fol- lowed by an isocratic elution at 26% solvent B (Fig. 2B, 0-14-5-4). The partial amino acid sequence of the peptide peak was determined (five turns of the sequenator) and revealed two sequences in equimolar ratios. The first, Asp- Leu-Leu-Thr-Pro, corresponds to amino acids 304-308 in the deduced 0 sequence. The second, Glu-Ile-Ser-Glu-Val, corresponds to amino acids 339-343 (Fig. 1). A peak con- sisting of only the first peptide sequence, which included Cys309 and ending at Ser311, has been independently iso- lated twice previously, and each time in single passes through the HPLC column. Utilizing similar elution conditions, this single peptide previously eluted at 26 min, rather than the 41 min reported here (Korneluk et al. 1986; Mahuran et al. 1988). The only difference in the two isolation techniques was that in the previous studies the protein had been reduced prior to digestion and HPLC separation. These data strongly suggest that these two peptides are covalently joined through a disulfide bond.

Whereas the predicted sequence of the first tryptic peptide would contain Cys309, the second sequence would include Glu339+ys359-Cys360)-Lys366 provided the Lys359-Cys360 peptide bond remained uncleaved by trypsin. Trypsin cleavage has been shown to take place more slowly when the basic residue is adjacent to an acidic residue or a cystine, and not at all when followed by Pro (reviewed in Smyth 1967). Thus, these data are consistent with a disulfide bond existing between Cys309 and Cys360 in the pro0 polypeptide and joining the Pb and 0, chains in the mature 0 subunit,

C 1 I 26 27 28 29

PERCENT SOLVENT B

"."a

TIME (min)

FIG. 2. Final reverse-phase HPLC purification of peak 8-14. Panel A shows its elution as /3-14-5 from a shallow gradient of 26-29% organic solvent B. Panel B shows its final base-line isola- tion by an isocratic elution at 26% solvent B as /3-14-5-4. The column was eluted isocratically at 1 mL/min for a span of 30 rnin.

placing the internal cleavage site Arg3 12-Gln-Asn3 14 in the pro0 chain near the amino terminus of a 50-residue loop structure (Fig. I).

Hex activity in COS cells transfected with the normal or mutant 0-cDNA constructs

As previously described, the wild-type pHexB43 directs the synthesis of a fully active, lysosomal Hex B in trans- fected cells. Furthermore, owing to the specificity of the solid state assay for human Hex, a 50- to 60-fold increase was observed in the specific activity that could be immobilized on the protein A - anti-human Hex B IgG complex (Brown et al. 1989; Neote et al. 1990; Brown and Mahuran 1991) (Table 1 ; compare bound COS( + ) with bound COS( - )). We next examined the amount of 4-MUG Hex activity produced by four independent transfections utilizing pHexB43-based cDNA constructs encoding various modifi- cations at the putative tripeptide clip site and the adjoining Lys315 (Arg312-Gln-Asn-Lys). These modifications included (i) deletion of all four residues, (ii) substitution of the four residues with the aligned residues from the human a chain (Gly-Ser-Glu-Pro), (iii) reintroduction of

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Fro. 3. Western blots of extracts from COS cells transfected with the four mutant cDNA constructs. Samples of each lysate con- taining equal amounts of 4-MUG activity (240 nmol/h) were denatured and reduced, and the protein was separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Hex-B-related bands were visualized using our polyclonal rabbit-anti-human Hex B IgG (Brown et al. 1989; Brown and Mahuran 1991). Untransfected COS cell lysates (COS(-)) were used as the negative control. The lysate from cells transfected with the normal pHexB43 plasmid (COS(+ )) was used as the positive control. (A) Deletion of the residues spanning the Pb-Pa cleavage site Arg312-Gln-Asn-Lys (ARQNK) or their replacement with the aligned residues from the human a subunit Gly- Ser-Glu-Pro (a (GSEP)) failed to produce a band corresponding to the mature 0, Mr. The band corresponding to 55 kDa represents the intact Pb+, polypeptide chain (lacking P,). (B) Replacement of P-Arg312 for the a-Gly residue Arg-Ser-Glu-Pro (Arg-a) or @-Lys315 for the a-Pro residue Gly-Ser-Glu-Lys (a-Lys), in the a-substitution cDNA construct, resulted in the regeneration of the cleavage as shown by the appearance of the mature Pa band. The presence of a small amount of the 55-kDa band, i.e., the PI,+, chain, demon- strates that the cleavage process was retarded by the presence of only one basic residue. Prestained protein Mr standards are also shown with their associated M, (as kilodaltons). The position of the immunoreactive bands corresponding to the mature 8, polypeptide and the intact @,+, chain are also shown.

Arg3 12 (Arg-Ser-Glu-Pro), (iv) reintroduction of Lys3 15 (Gly-Ser-Glu-Lys), and (v) replacing Arg312 with Gln in the original 0 sequence (Gln-Gln-Asn-Lys). In all the experi- ments, the specific activity of the immobilized mutant Hex B's were greater than or equal to that of the wild-type COS( +) (Table 1; COS constructs i-v). Furthermore, activ- ity values for the unbound enzyme (endogenous COS Hex) did not vary significantly from that of the negative COS cell control value (Table 1; COS(-)), indicating that none of the mutations we produced adversely affected the reactivity of our anti-human Hex antiserum with the translated 0 protein. Human Hex B activity measurements of the cell culture media demonstrated that the percentage of enzyme secreted by the COS cells transfected with the mutant cDNA constructs was less than or equal to that of cells transfected with COS(+), the control pHexB43 (Table 1; compare percent secretion of the "COS mutant constructs" to COS(+)). Thus, the mutations had no detrimental effect on the specific activity or intracellular retention of the translated enzyme.

Western blot analysis of the protein synthesized from the 0-cDNA constructs

Western blot analysis allowed us to examine the effect of the various mutations on the normal processing of the @ subunit from the Hex B produced by transfected COS cells. Since our antibody primarily recognizes antigens on the 0, chain (Brown et al. 1989; Brown and Mahuran 1991), three possible M, values (as determined by polyacryl- amide gel electrophoresis in sodium dodecyl sulfate of

reduced and denatured protein samples) could be expected based on the degree of proP processing: (i) unprocessed proP precursor, i.e., Pp+ b + a r 65 kDa; (ii) cleavage of the amino- terminal 0, chain, but not the Pb+, chains, 55 kDa; or (iii) cleavage of the fib (or /3p+b) polypeptide from the 0, chain, 28 kDa.

Western blots of COS cell extracts from the five trans- fections with cDNA constructs encoding modifications in the clip site (described above) were compared with that of an extract from cells transfected with the wild-type pHexB43. The pattern of the immunoreactive protein band from reduced and denatured cell lysates transfected with the wild-type pHexB43 COS( + ) showed the expected mature, 28-kDa Pa band, confirming proper lysosomal internaliza- tion (Brown et al. 1989; Neote et al. 1990; Brown and Mahuran 1991). The 0, band was not found in reduced lysates from cells transfected with the deletion construct (Fig. 3A, ARQNK) or the a substitution cDNA construct (Fig. 3A, a (GSEP)). The 55-kDa immunoreactive band detected in these lysates corresponds to Pb+,, a partially processed /3 form lacking the 6, chain (Fig. 3A). These results suggest that a specific sequence or residue(s) unique to the original 0-tetrapeptide is necessary for the proteolysis to occur.

Replacement of the nucleotides encoding P-Arg312 or Lys315 in the a-substitution construct, resulted in the regeneration of the cleavage event in transfected cells, i.e., the appearance of the mature 8, band in reduced cell lysates (Fig. 3B, Arg-a and a-Lys, respectively). These data indicate that the internal cleavage most likely occurs in vivo

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SAGHERIAN ET AL. 345

at both basic amino acids. However, the presence of observ- able levels of uncleaved Pb+a chains (Fig. 3) suggest that proteolytic processing has been slowed either by the presence of only one basic amino acid or by the presence of the surrounding a sequence. To differentiate between these two possibilities a construct was prepared that contained the original /3 sequence, but with neutral Gln substituted for basic Arg312 (QQNK). Examination by Western blot anal- yses of COS cells transfected with this construct gave virtu- ally identical results to that seen for the wild-type construct (Fig. 4). Thus, whereas cleavage at either basic residue is not absolutely dependent on the identities of the surrounding amino acids, the speed of proteolytic processing is influenced by these residues.

Substrate specifcity and heat stability of the clip-site mutants

To determine if biochemical properties unique to the subunit of Hex B (i.e., substrate specificity and heat

stability) were in any way linked to prop's unique processing event, the enzyme produced by the a substitution construct (Pb-Arg-Gln-Asn-Lys-P, - /3b-Gly-Ser-Glu-Pro-P,) which is not processed (Fig. 3A, a (GSEP)) was tested for an increase in 4-MUGS (an a-specific substrate) activity. The results indicated that the substrate specificity of the mutated Hex B was unaltered; i.e., it still could not hydrolyze the sulfated artificial substrate (data not shown). We next examined the heat stability of the mutant Hex B enzymes at 60°C. The human Hex B activity in lysates from cells transfected with wild-type pHexB43 had a t lIz similar to that of purified placental Hex B (Table 1). The Hex B synthesized by all but the deletion construct had similar heat stability. The deletion construct produced an enzyme with a tlI2 about 25% that of the wild-type enzyme (see Table 1, AR312QNK). Since both the enzymes produced by the deletion and the a-substitution cDNA constructs do not undergo the internal cleavage of the Cys309-Cys360 loop structure, these stability experiments demonstrate that neither the lack of cleavage in the proa chain nor the difference in its primary sequence in the region is responsible for the difference in heat stability between Hex A and Hex B.

Discussion In this report we demonstrate that the &-Pa cleavage site

is contained within an exposed Cys309-Cys360 loop structure whose length, 49-50 residues, has been evolutionarily con- served from Dictyostelium discoideum (Fig. 1). The conser- vation in length suggests that nearly the full complement of 50 residues may be necessary for the formation of a stable disulfide bond in the proa and prop subunits. This hypoth- esis would explain why the deletion construct was the only one to produce an enzyme that was less heat stable than the wild-type Hex B (Table 1).

We also present further evidence for the location and mechanism of the internal cleavage (1) of the prop chain. Our data suggest that two endoproteolytic events take place in a trypsin-like manner, except that hydrolysis occurs on the amino rather than the carboxyl side of basic residues, i.e., Pb-Ser 1 Arg312-Gln-Asn3 141 Lys-pa. Since the loss of Lys315 from the N-terminus of the Pa chain has been shown to be a later processing event (Quon et al. 1989; Mahuran 1990), it could be suggested that a more conven- tional cleavage occurs initially at the C-terminus of Arg3 12.

FIG. 4. Western blots of extracts from COS cells transfected with a mutant P-cDNA construct encoding an Arg312-Gln substi- tution. The sample of mutant lysate contained twice the amount of 4-MUG activity as the positive control COS(+) to determine if any intact chains were present, as in Fig. 3B.

The removal of Arg312 and of Gln-Asn314, as well as Lys315, could then proceed through carboxyl and amino peptidase digestion, respectively. If these events occurred, they would have to be rapid as no such N-terminal inter- mediates were found to be generated in biosynthetic studies of the Hex isozymes in human fibroblasts; i.e., only Lys315 was found as the N-terminus of the 28-kDa Pa chain in pulse-chase experiments (Quon et al. 1989). Furthermore, the demonstration that cleavage occurs in fl proteins lacking either Lys315 or Arg3 12 (i.e., either Arg3 12-Ser-Glu-Pro (Arg-a), or Gly312-Ser-Glu-Lys (a-Lys) or Gln312-Gln-Asn- Lys), but not in P proteins lacking both Arg312 and Lys315 (i.e., a (GJ12SEP) and AR312QNK), strongly supports the hypothesis that there are two endopeptidase sites in this small domain. These data also indicate that the identities of the residues flanking Arg312 or Lys315 do not play a critical role in the recognition of the site by the endopeptidase.

The lysosome contains five well-characterized endopep- tidases: cathepsins B, D, H, L, and S. Previous studies have demonstrated that at least two proteases are involved in the maturation of the prop chain, and that the protease gener- ating the pb and pa polypeptides has most likely not been characterized (reviewed in Hasilik 1992). Whereas processing of cathepsin D is normal, the Hex Pb-Pa cleavage event does not occur in human monocyte line U937, because of its lack of a specific uncharacterized protease (Gupta et al. 1984). Secondly, we found that pHexB43-transfected cells grown in media containing pepstatin, an aspartyl protease inhibitor, produced fully processed Pa chains (data not shown). Finally, we (Brown and Mahuran 1991) demon- strated that the cleavage of the amino-terminal 0, chain

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346 BIOCHEM. CELL BIOL. VOL. 71, 1993

Surface Structure

160 Beta Turn 12 0 Coil

80 Beta Sheet

40 Alpha Helix

0 GQKDLLTPCYSRQNKLDSFGPINPTLNTTYSFLTTFFKEISEVFPDQFIHLGGDEVEFKCWESNPKIQD

I - I FIG. 5. Graphical output from the program SURFACE (Emini et al. 1985) (left y-axis) overlaid with the results from a secondary

STRUCTURE prediction program as calculated by the Garnier technique (Garnier et al. 1978) (right y-axis). In the latter case the most likely structure is indicated by the height of a line (at one of four possible positions corresponding to the four possible structures) over the corresponding residues (x-axis). The analyses were carried out using GENE WORKS^^ (IntelliGenetics, Inc.). The cleavage site is double underlined and the glycosylation site, the primary substrate for the lysosomal protein phosphotransferase (Sonderfeld-Fresko and Proia 1989), is underlined.

from Pb, but not the Pb-Pa cleavage, is inhibited in pHexB43-transfected COS cells by including leupeptin, an inhibitor of thiol-dependent cathepsins (e.g., cathepsins B, H, L and S) (Barrett and Kirschke 1981) and serine proteases (e.g., trypsin and lysosomal acrosin) (Umezawa 1976) in the growth media. Thus, none of the previously characterized endopeptidases appear to be involved in &,-Pa cleavage.

The protein domain, assumed to be common to all lysosomal proteins, that is recognized by the phosphotrans- ferase has yet to be identified (reviewed in Kornfeld 1990). However, recent evidence has suggested that hydrophilic surface areas possibly containing a Lys residue in close spatial proximity to an oligosaccharide substrate may play a role (Baranski et al. 1990, 1992; Cantor et al. 1992). The fourth glycosylation site in the pro0 chain at Asn327 (Figs. 1 and 5) is the primary substrate for the lysosomal enzyme phosphotransferase (Sonderfeld-Fresko and Proia 1989). The predictive data from the SURFACE analysis (Emini et al. 1985) (Fig. 5) indicates that both the Lys315 and Asn327 reside in exposed areas of the protein and are predicted to be contained within &turn structures by the Garnier tech- nique (Garnier et al. 1978) (Fig. 5). Thus, the RQNK315 0-cleavage site is a candidate recognition site in the propo- lypeptide for the phosphotransferase. However, our data clearly demonstrate that neither the replacement of Lys315 or the removal of the entire cleavage site increases the per- centage of human Hex secreted by transfected cells (Table 1). Therefore, this area can be eliminated from the continuing search for the phosphotransferase recognition site in Hex.

The hydrophilic nature and 0-turn secondary structure that is predicted to be the environment of the Pb-Pa cleavage site indicate that the inclusion in an exposed area may be required for the recognition of Arg and Lys residues by the endopeptidase. Such a requirement would explain why other nearby basic residues are not hydrolyzed. For example, Lys338 and Lys359 (Fig. 1) are also located in the loop structure, but in far less hydrophilic regions, and may be contained in a helices (Fig. 5). It could also help to explain why the speed of processing was slowed in the a-Lys and Arg-a constructs (Fig. 3). The substituted a sequence is

predicted to be somewhat less hydrophilic than the normal 0 sequence (data not shown).

Our data are consistent with the idea that the partially degraded mature Hex isozymes are the resistant, but still functional products of the proteins' adaptation to the harsh lysosomal environment. We have demonstrated in this report that at least some of the residues removed from the pro0 chain in the lysosome are not critical to the intracellular targeting, substrate specificity, or stability of the native isozymes. However, these residues appear to have been necessary for the initial folding and disulfide bond formation of the pro0 polypeptide in the endoplasmic reticulum.

Acknowledgments We wish to thank Ms. A. Leung and Mr. Yong-Jian Wu

for their excellent technical assistance. The amino acid sequencing and oligonucleotide syntheses were done by The Hospital For Sick Children Biotechnology Service. This work was supported by the Medical Research Council of Canada grant MA-10435.

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