©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lysosomal Protective Protein/Cathepsin A
ROLE OF THE ``LINKER'' DOMAIN IN CATALYTIC ACTIVATION (*)

(Received for publication, July 5, 1995; and in revised form, August 30, 1995 )

Erik J. Bonten Niels J. Galjart (1) Rob Willemsen (2) Magda Usmany (3) Just M. Vlak (3) Alessandra d'Azzo (§)

From the  (1)Department of Genetics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, the Departments of Cell Biology and (2)Clinical Genetics, Erasmus University, Rotterdam 3000 DR, The Netherlands, and the (3)Department of Virology, Agricultural University, Wageningen 6700 EM, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Lysosomal protective protein/cathepsin A is a serine carboxypeptidase that forms a complex with beta-galactosidase and neuraminidase. The enzyme is synthesized as a 54-kDa precursor/zymogen and processed into a catalytically active 32- and 20-kDa two-chain form. We have expressed in baculovirus-infected insect cells the human one-chain precursor as well as the two separate subunits in order to establish the mode of catalytic activation of the zymogen and the assembly and activation of the two subunits. Infected insect cells synthesize large quantities of the exogenous proteins, which are glycosylated and secreted but not processed. Co-expression of the two subunits results in their assembly into a two-chain form of 34- and 20-kDa with negligible enzymatic activity. Limited proteolysis with trypsin of the 54-kDa precursor and the reconstituted 34- and 20-kDa form gives rise to a fully active 32- and 20-kDa product. These results enabled us to map the sites of proteolytic cleavage needed for full activation of the cathepsin A zymogen. They further indicate that the 34- and 20-kDa form is a transient processing intermediate that is converted into a mature and active enzyme by removal of a 2-kDa ``linker'' peptide from the COOH terminus of the 34-kDa subunit.


INTRODUCTION

A primary defect of lysosomal protective protein/cathepsin A (PPCA) (^1)in humans causes the metabolic storage disorder galactosialidosis ((1) ; reviewed in (2) ). This disease is characterized by severely reduced activities of the enzymes beta-D-galactosidase and N-acetyl-alpha-neuraminidase, secondary to absent or abnormal PPCA. The reason for the additional combined deficiency relates to one of the functions of PPCA protein, which is to associate with and protect the two glycosidases, modulating their activity and stability in lysosomes(1, 3, 4, 5) . The primary structures of human, mouse, and chicken PPCAs are highly conserved (6, 7, 8) and bear homology to yeast and plant serine carboxypeptidases ((6) ; for reviews see Refs. 9 and 10). Mammalian PPCAs have cathepsin A activity at lysosomal pH but maintain a deamidase/esterase activity at neutral pH(8, 11) . Furthermore, the human enzyme, purified from platelets and lymphocytes, has been shown to function both in in vitro and in vivo assays on the inactivation of selected neuropeptides, like substance P, oxytocin, and endothelin I(11, 12, 13) .

PPCA is synthesized as a 54-kDa precursor that is glycosylated on two Asn residues and is enzymatically inactive(1, 6, 8) . The precursor dimerizes at neutral pH shortly after synthesis and is likely to be transported as a dimer to the lysosomes(14) . Once in the acidic lysosomal environment, the zymogen is cleaved into an enzymatically active 32- and 20-kDa two-chain form. However, the events involved in proteolytic activation have not been established until now. Here we have used baculovirus (BV)-expressed human precursor and two separate subunits to map the sites of cleavage and processing of human PPCA zymogen. We have identified a ``linker'' domain in the precursor molecule, located at the COOH terminus of the large subunit, which needs to be removed for full catalytic activation of the enzyme.


EXPERIMENTAL PROCEDURES

Plasmid Constructs

AcMNPV transfer plasmids pJR2 and pBC3 are derivatives of plasmid pAc373, which includes the entire polyhedrin gene(15) . They both contain a polylinker with multiple cloning sites, inserted directly 3` of the polyhedrin promoter. In pJR2 the polylinker substitutes a 33-nucleotide deletion spanning the ATG, whereas in pBC3 only the ATG codon is mutated to ACG. Full-length human PPCA cDNA, HPP54(6) , and the two deletion cDNA mutants, HPP32(Delta20) and HPP20(Delta32)(8) , were subcloned either in pJR2 or pBC3 as EcoRI fragments, using standard procedures(16) . The HPP20(Delta32) deletion mutant was tagged with the human PPCA signal sequence, as reported earlier(8) . All cDNA fragments were engineered to have short 3`- and 5`-untranslated regions (<10 base pairs).

Generation of Recombinant Baculoviruses

Spodoptera frugiperda insect cells (IPLB-SF21) were cultured in monolayers at 27 °C in TNM-FH medium (17) supplemented with 10% fetal bovine serum and antibiotics (complete medium). Wild-type AcMNPV virus strain E2 (18) and recombinant baculoviruses were propagated on confluent monolayers of Sf21 cells. Recombinant baculoviruses were generated by co-transfecting Sf21 cells with 1 µg of wild-type AcMNPV DNA and 10 µg of plasmid DNA as described(19) . They were selected and purified by sequential plaque assays and verified by Southern blot analysis(19) . Large quantities of inoculum were produced by infection of insect cells at 25-50% confluence with recombinant virus at a multiplicity of infection of < 1 plaque-forming unit/cell. After 3-6 days at 27 °C, when all cells appeared infected, the medium was harvested and centrifuged for 5 min at 1000 rpm to remove detached cells. The titre of the inoculum was determined by plaque assay analysis.

Metabolic Labeling of Infected Cells

For biosynthetic labeling studies, Sf21 cells were seeded in 6-well plates and grown until 80-90% confluence. Cells were infected with baculoviruses at a multiplicity of infection of 5-10 plaque-forming units/cell and radiolabeled for 4 h with 50 µCi/ml [S]methionine (Amersham Corp.). Immunoprecipitations were carried out with anti-human PPCA precursor antibodies (anti-54) (8) and formalin-fixed Staphylococcus aureus cells (Immunoprecipitin, BRL) as reported earlier(8, 20) . Immunoprecipitated proteins were resolved on 12.5% SDS-polyacrylamide gels under reducing or nonreducing conditions (21) and visualized by fluorography of gels soaked in Amplify (Amersham Corp.).

Immunoelectron Microscopy

Sf21 cells infected with recombinant baculoviruses were fixed two days postinfection in 0.1 M phosphate buffer, pH 7.3, 1% acrolein, and 0.4% glutaraldehyde. Further embedding in gelatin, preparation for ultracryotomy, and methods for immunoelectron microscopy were as reported earlier(22) . Ultrathin sections were probed with antibodies (anti-32) raised against the denatured 32-kDa chain of human PPCA (6) .

Development of Anti-peptide Antibodies

A 15-amino acid peptide (NH(2)-Cys-Met-Trp-His-Gln-Ala-Leu-Leu-Arg-Ser-Gly-Asp-Lys-Val-Arg-COOH) based on the COOH-terminal sequence of the 34-kDa subunit (amino acids 285-298) (6) was synthesized on a peptide synthesizer (Applied Biosystems) and covalently coupled via the NH(2)-terminal Cys residue to the carrier protein Keyhole Limpet Hemocyanin using the Imjet-activated immunogen conjugation kit as recommended by the manufacturer (Pierce). Polyclonal antibodies (anti-pep) were raised in rabbits against the conjugated product and tested on immunoblots or immunoprecipitations of BV-produced proteins.

Limited Proteolysis with Trypsin

Infections with baculovirus constructs were performed as described above. Medium samples were concentrated 5-20-fold by ammonium sulfate precipitation and desalted on a Sephadex G50 column(20) . Aliquots of 15 µl of medium concentrates were diluted to 200 µl with 10 mM sodium phosphate buffer, pH 6.8, and 1 mg/ml bovine serum albumin. Samples were subjected to limited proteolysis with 1 µg of trypsin (Sigma) for increasing periods of time as described by Galjart et al.(8) . Reactions were stopped by the addition of 3 µg of bovine pancreas trypsin inhibitor (Sigma). Aliquots of each reaction mixture (10 µl) were assayed for cathepsin A activity using the N-blocked dipeptide benzyloxycarbonyl-phenylalanyl-alanine as described by Galjart et al.(8) . 20-µl aliquots of trypsin-digested proteins were resolved on SDS-polyacrylamide gels and transferred from gels to Immobilon polyvinylidene difluoride membranes (Millipore Corp.) using a semi-dry blotter (W. E. P. Company). Blots were incubated for at least 12 h in blocking buffer (0.01 M Tris-buffered saline, pH 8.0, 0.05% Tween 20, and 3% (w/v) bovine serum albumin) and subsequently probed with anti-54 or anti-pep antibodies followed by alkaline phosphatase conjugate anti-rabbit IgG second antibodies (Sigma). Proteins were stained using the colorimetric substrate for alkaline phosphatase (Sigma). For the DFP-binding assay, 40-µl aliquots were incubated for 1 h at room temperature with 1 µCi of [^3H]DFP as described earlier(8) . Radiolabeled proteins were immunoprecipitated with anti-54 antibodies and resolved by SDS-polyacrylamide gel electrophoresis and fluorography.


RESULTS

SF21 insect cells were infected with recombinant baculoviruses AcHPP54, AcHPP32, and AcHPP20 separately or co-infected with AcHPP32 and AcHPP20. Metabolic labeling and immunoprecipitation analysis showed that PPCA precursor was synthesized in large quantities and efficiently secreted but was not or poorly processed to the mature two-chain product (Fig. 1, lanes 1 and 5). Its estimated molecular mass of 54 kDa was similar to wild-type precursor from human cultured fibroblasts(1) . Trace amounts of low molecular weight polypeptides, visible both intra- and extracellularly, were products of aspecific proteolysis, because pulse-chase experiments showed no time-dependent conversion of the precursor into mature protein (not shown). Single infections with AcHPP32 and AcHPP20 resulted in the production of truncated polypeptides with molecular masses of 34, 20, and 18 kDa, respectively (Fig. 1, lanes 2 and 3). Only the former, which was 2 kDa larger than the corresponding wild-type subunit, was secreted to some extent (Fig. 1, lane 6). The two chains of 20 and 18 kDa (Fig. 1, lane 3) were different glycosylation forms of the small subunit. Under nonreducing conditions the secreted large chain migrated as a doublet, suggesting that within this polypeptide partial and/or different intrachain disulfide bridges may have formed (Fig. 1, lane 10). Co-infected cells synthesized a large subunit of 34 kDa and three small subunits of 20, 19.5, and 18 kDa, respectively. The intermediate 19.5-kDa species was unique for co-infected cells (compare lanes 3 and 4) and was the only one of the small subunits found in the medium (Fig. 1, lanes 8 and 12). Co-expressed polypeptides were immunoprecipitated in larger quantities both intra- and extracellularly, compared with single infections and under nonreducing conditions were resolved as one product of 54 kDa, similar in size to the wild-type precursor (Fig. 1, lanes 9 and 12). These results suggest an early intracellular association of the two separately synthesized polypeptides, which are secreted in an associated state. Although they assemble, the 34-kDa subunit remains 2 kDa bigger (Fig. 1, lane 8) and is not further processed to the mature size.


Figure 1: Metabolic labeling of PPCA precursor and separate subunits in insect cells. Sf21 cells were either infected with baculovirus construct AcHpp54 (54), AcHpp32 (32), or AcHpp20 (20), or co-infected with AcHpp32 and AcHpp20 (32/20). 2 days after infection newly synthesized proteins were labeled with [S]methionine. Labeled proteins were immunoprecipitated from cells and media using anti-54 antibodies. Proteins were separated by SDS-polyacrylamide gel electrophoresis under reducing (lanes 1-8) or nonreducing (lanes 9-12) conditions and visualized by fluorography. Exposure time was 24 h. Molecular sizes are indicated. DTT, dithiothreitol.



To determine whether the subcellular localization of over-expressed proteins could account for the lack of maturation, singly infected or co-infected cells were analyzed with electron microscopy. Immunostaining of ultrathin sections demonstrated an intracellular distribution that was similar for the different over-expressed proteins. Extensive gold labeling was restricted to structures in the cytoplasm corresponding to swollen cisternae of the endoplasmic reticulum (Fig. 2A) and the Golgi complex (Fig. 2B). Other subcellular organelles, including multivesicular bodies and fibrous structures, usually observed in insect cells infected with either wild-type or recombinant viruses(23, 24) , were totally devoid of gold particles. It appears therefore that none of the BV-expressed proteins reach a lysosome-like compartment, where proteolytic processing should occur.


Figure 2: Subcellular localization of BV-expressed proteins. Sf21 cells were infected with AcHPP54, AcHPP32, or AcHPP54 + AcHPP32 and prepared for immunoelectron microscopy 48 h after infection. Cryosections were incubated with anti-32 antibodies followed by goat anti-rabbit IgG gold labeling. A, in all three cases the over-expressed proteins were localized in swollen cisternae of the endoplasmic reticulum (R), which have the appearance of large vacuoles. B, gold particles were also clearly present in the Golgi complex (G). n, nucleus; F, fibrous structures; MVB, multivesicular bodies. The magnifications were: 38,000times for A and 79,000times for B.



The amino terminus of the 20-kDa chain, isolated from human placenta and human platelets(6, 11) , starts with Met-299, which is preceded in the amino acid sequence of the precursor by a conserved arginine. This residue likely represents the site of initial cleavage. Circumstantial evidence has, however, indicated that complete maturation of the precursor may require more proteolytic steps that could occur at Arg-284, Arg-292, or Lys-296. Here we have monitored the processing and catalytic activation of human PPCA precursor and reconstituted subunits by digesting BV-derived secreted proteins with trypsin. To ascertain the occurrence of sequential processing steps, we have used a polyvalent rabbit antibody (anti-pep) raised against a peptide of 15-amino acids, 14 of which are derived from the COOH terminus of the large subunit. Concentrated medium samples containing either the precursor or the 34- and 20-kDa associated protein were incubated with a fixed amount of trypsin for increasing periods of time, and each reaction was stopped by the addition of trypsin inhibitor. Aliquots of each sample were tested on Western blots immunostained with anti-54 and anti-pep antibodies and were assayed for cathepsin A activity. As seen in Fig. 3(upper left panel), after 0.5 min of incubation with trypsin, part of the one-chain precursor was cleaved into a two-chain product of 34 and 20 kDa. Between 2 and 5 min, the 34-kDa form was gradually converted into a 32-kDa derivative, and complete maturation was achieved after 10 min. In contrast, the size of the 20-kDa chain did not vary. Prolonged digestion periods (15 and 30 min) led to aspecific degradation and lower yield of both subunits. Using anti-pep antibodies, only the 54- and 34-kDa polypeptides were detected, indicating that the COOH-terminal peptide of the 34-kDa species was lost upon conversion to the 32-kDa form (Fig. 3, middle left panel). Step-wise maturation of the precursor into a fully processed product was paralleled by a clear increase in cathepsin A activity, which was maximal after 15 min of digestion (Fig. 3, lower left panel). Similar maturation steps were observed for the reconstituted 34- and 20-kDa protein. Upon trypsin cleavage, the large chain was again converted from a 34- to a 32-kDa product, whereas the size of the small subunit did not change (Fig. 3, upper and middle right panels). However, the amount of both polypeptides, as detected on immunoblots, was significantly less than for wild-type precursor, resulting in an overall reduced cathepsin A activity. This was probably due to both a lower secretion of the reconstituted two-chain protein as well as a reduced stability. However, also in this case a clear increase in enzymatic activity was measured after digestion (Fig. 3, lower right panel).


Figure 3: Limited proteolysis with trypsin of 54-kDa precursor and 34- and 20-kDa reconstituted two-chain protein. Aliquots of medium concentrates containing the 54-kDa precursor and 34- and 20-kDa associated protein were incubated at 37 °C with 1 µg of trypsin in the presence of bovine serum albumin (1 mg/ml) for the indicated periods of time. Reactions were stopped with 3 µg of trypsin inhibitor. Samples in lanes 1 and 9 were untreated. At time 0 (lanes 2 and 10), the samples were treated with trypsin inhibitor prior to the addition of trypsin. A portion of each sample was separated by SDS-polyacrylamide gel electrophoresis, followed by electroblotting and immunostaining with anti-54 and anti-pep antibodies. Cathepsin A activity toward the acylated dipeptide benzyloxycarbonyl-phenylalanyl-alanine was measured in each aliquot. One milliunit (mU) of activity is defined as the enzyme activity that releases one nanomole of alanine/minute.



Catalytic activation of precursor and reconstituted products was confirmed by the ability of trypsin-processed proteins to bind the serine protease inhibitor DFP. As shown in Fig. 4(lanes 1-6), only the mature 32-kDa subunit, generated after trypsin digestion of the precursor, was recognized by the radiolabeled inhibitor. The 34-kDa form was apparently unable to bind DFP, although it was present at 0.5-5 min digestion time points (see Fig. 3). The inability to bind the inhibitor was particularly evident in the case of the undigested 34- and 20-kDa protein (lane 7), which showed no radioactive signal, confirming that it has only marginal enzymatic activity. However, this associated form was particularly susceptible to proteolytic cleavage, because immediately after addition of trypsin (time 0, lane 8), a substantial conversion to the 32-kDa product took place. For both wild-type precursor and reconstituted protein, the highest levels of cathepsin A activity were measured at digestion time points in which maximal binding was observed (Fig. 4, lanes 3-5 and 9-10). As seen in the previous experiment, the overall amount of reconstituted and digested two-chain product was again considerably lower than trypsin-cleaved precursor. All together these data point to the 34- and 20-kDa product as being a processing intermediate, transiently occurring during proteolytic maturation of PPCA precursor. This process is probably mediated by a trypsin-like protease. Removal of a COOH-terminal peptide from the 34-kDa subunit is essential for catalytic activation.


Figure 4: Binding of serine carboxypeptidase inhibitor DFP to trypsin digested 54 kDa precursor and reconstituted two-chain protein. Trypsin digestions of precursor and 34- and 20-kDa two-chain product were performed as described in the legend to Fig. 3. 1 µCi of [^3H]DFP was added to 40 µl of trypsin digest, followed by immunoprecipitation with anti-54 antibodies. Immunoprecipitated proteins were separated on SDS-polyacrylamide gels under reducing conditions and visualized by fluorography. Exposure times were 2 weeks for lanes 1-6 and 3 months for lanes 7-12. Molecular sizes are indicated. Cathepsin A activity was measured as described in the legend to Fig. 3.




DISCUSSION

PPCA is a lysosomal serine protease with pleiotropic biological properties. It binds in its normal state to the enzymes beta-galactosidase and neuraminidase, rendering them stable and active in lysosomes; it also hydrolyzes as carboxypeptidase and/or deamidase/esterase a variety of bioactive peptides, depending upon the pH conditions used in the assay. When deficient or defective in humans, it causes the lysosomal storage disease galactosialidosis. Because its protective and catalytic functions are distinct(8) , we and others have postulated a role for PPCA in the local inactivation of selected neuropeptides, with or without the aid of the two glycosidases(8, 11, 12, 13) . The protein is synthesized in mammalian tissues and cultured cells as a one-chain precursor that is enzymatically inactive. Proteolytic conversion to a disulfide-linked two chain product triggers catalytic activation. To investigate the process of zymogen activation and to assess the capacity of the separately synthesized subunits to assemble into an active enzyme, we have used the baculovirus system (for review see (25) and references therein) to express human PPCA in insect cells, either as one-chain precursor or as two separate subunits.

BV-encoded proteins are synthesized in large amounts and transported from the endoplasmic reticulum to the Golgi complex but do not seem to be targeted to a lysosome-like organelle. It is conceivable that this transport step either requires a recognition marker on lysosomal proteins specific for insect cells or the process is saturated by the high concentration of newly synthesized proteins. The subcellular distribution of both PPCA precursor and reconstituted subunits could however explain their inadequate intracellular maturation, which may depend on a lysosome-associated protease. Although it is known that the degree of proteolytic processing varies among heterologous proteins expressed with the baculovirus system(25) , identical results were obtained by us with mouse PPCA and human beta-galactosidase expressed in insect cells (^2)and by others with human beta-galactosidase, beta-glucosidase, and beta-hexosaminidase (26, 27, 28, 29) . Of the three separately synthesized proteins, mainly the wild-type precursor is secreted in a large quantity. Interestingly, however, the co-expressed subunits are able to form interchain disulfide bridges and are secreted only in the associated state. They evidently require conformational changes for their secretion, acquired only when both domains are present. Using baculovirus co-expression vectors, other investigators have demonstrated appropriate formation of functional immunoglobulin heterodimers, which are efficiently secreted, and of disulfide-bridged interleukin 5 homodimers(30, 31) .

The focus of the experimental work presented here is the process of enzymatic activation of human PPCA zymogen, which enabled us to map the linker domain between the two chains, whose removal is required for activation. Both wild-type precursor and reconstituted two-chain form can be subjected to partial proteolysis with trypsin, and the cleaved products have maximal cathepsin A activity. Our results suggest that the in vitro activation of BV-produced precursor mimics its in vivo processing taking place in at least two steps: an endoproteolytic cleavage resulting in a transient intermediate of 34- and 20-kDa, followed by trimming of the last 14 amino acids at the COOH terminus of the large chain. Furthermore, we provide evidence that the 34- and 20-kDa reconstituted product is probably identical to the partially processed intermediate, which is a naturally occurring catalytically inactive form. A similar processing pattern was observed earlier for PPCA precursor over-expressed in COS-1 cells(8, 14) . In this case the 34- and 20-kDa intermediate is seen only transiently, indicating that its half-life may be too short and its quantity too low to be detected in normally expressing cultured fibroblasts. There is an interesting analogy between the processing and activation of PPCA and that of other serine carboxypeptidases. This family of proteases comprises single- and two-chain enzymes that are present in different species, ranging from yeast to fungi, plants, and humans(9, 10) . Some of the plant peptidases have been purified in an active form composed of two chains that have been sequenced at their NH(2) termini(32) . In the case of barley carboxypeptidase I, the two mature subunits of the enzyme originate from a single-chain precursor that in addition contains a stretch of amino acids separating the two chains(33) . This closely resembles the processing of PPCA zymogen and provides another verified example in a homologous carboxypeptidase of COOH-terminal trimming after the initial endoproteolytic step. The analogy in maturation events might indicate a similar function for the linker domain in the plant carboxypeptidase and PPCA precursors, namely to keep these forms in an inactive state. In yeast carboxypeptidase Y such an inactivation function resides within its propeptide segment, which must obstruct the access of a substrate to the preformed active site(34) . Limited proteolysis with trypsin removes this inactivating segment and exposes the active site. Removal of the linker domain during maturation of the PPCA zymogen may induce major conformational changes on the protein necessary to uncover its active site. Alternatively, this peptide may only mask the active site on an already correctly folded polypeptide. These hypotheses are currently verified on the basis of the three-dimensional structure of the uncleaved precursor molecule.


FOOTNOTES

*
These studies were supported in part by the National Institutes of Health Cancer Center Support CORE Grant P30-CA21765, the American Lebanese Syrian Associated Charities, and the Foundation of Clinical Genetics at the Erasmus University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Genetics, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Fax: 901-526-2907.

(^1)
The abbreviations used are: PPCA, protective protein/cathepsin A; BV, baculovirus; DFP, diisopropyl fluorophosphate.

(^2)
Erik J. Bonten, unpublished data.


ACKNOWLEDGEMENTS

We are indebted to Prof. Hans Galjaard (Erasmus University, Rotterdam) for support. We thank Dr. Gerard Grosveld for stimulating discussions and critical reading of the manuscript. We gratefully acknowledge the help of Dr. Richard Proia (NIDDK, NIH, Bethesda, MD), who has synthesized for us the synthetic peptide used in these studies. We also thank Peggy Burdick for editing the manuscript.


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