(Received for publication, October 3, 1995; and in revised form, January 24, 1996)
From the
Leishmanolysin (EC 3.4.24.36) (gp63) is a HEXXH
metalloprotease, encoded by multicopied genes in Leishmania and implicated in the infectivity of these parasitic protozoa. We
examined posttranslational regulation of gp63 expression by
site-specific mutagenesis of the predicted catalytic/zinc-binding sites
in the HEXXH motif, the potential sites of N-glycosylation and glycosyl phosphatidylinositol addition.
Mutant and wild-type genes were cloned into a Leishmania-specific vector for transfecting a deficient
variant, which produced gp63
20-fold less than wild-type cells.
The selective conditions chosen fully restored this deficiency in
transfectants with the wild-type gene. Under these conditions, all
transfectants were found comparable in both the plasmid copy number per
cell and elevation of gp63 transcripts. Mutant and wild-type products
in the transfectants were then compared quantitatively and
qualitatively by specific immunologic and protease assays. The results
indicate the following. 1) Glu-265 in the HEXXH motif is
indispensable for the catalytic activity of gp63. The propeptide of the
inactive mutant products was cleaved, suggestive of a
non-intramolecular event. 2) Substitution of either His residue in
HEXXH leads to apparent intracellular degradation of the
mutant products, pointing to a role for zinc binding in in vivo stability of gp63. 3) The three potential sites of N-glycosylation at Asn-300, Asn-407, and Asn-534 are all
utilized and contribute to intracellular stability of gp63. 4)
Substitution of Asn-577 causes release of all mutant products,
indicative of its specificity as a glycosyl phosphatidylinositol
addition site for membrane anchoring of gp63. It is suggested that
expression of gp63 as a functional protease is regulated by these
posttranslational modification pathways.
Some metalloproteinase families, e.g. thermolysin, astacin, serratia, matrixin, and snake venom, share the signature sequence of HEXXH as a conserved zinc-binding/catalytic motif(1) . Leishmanolysin (EC 3.4.24.36) (2) is such a zinc protease (gp63) on the surface of the trypanosomatid protozoa, Leishmania(3, 4, 5) . They live extracellularly as promastigotes in the gut of the insect vector and intralysosomally parasitize macrophages as amastigotes in the mammalian host, causing human leishmaniasis. gp63 has been suggested to function in multiple steps of the infection(6) , i.e. their entry into macrophages (see (7, 8, 9, 10, 11) ) and intralysosomal survival(5, 12) . Leishmanolysin thus appears to differ functionally from other HEXXH metalloproteinase families.
Different Leishmania gp63 genes are highly sequence-conserved (13, 14, 15, 16, 17) and clustered into two to three tandem repeats usually in a single chromosome(17, 18, 19, 20) . gp63 is abundantly expressed in promastigotes, apparently due to polycistronic transcription of these repetitive genes followed by their trans-splicing into multiple copies of functional mRNAs. In Leishmania chagasi, the growth phase-dependent expression of gp63 appears to result from selective degradation of the off-phase mRNAs rather than new transcription of the in-phase mRNAs(21) . Thus, the specific expression of gp63 is apparently regulated posttranscriptionally, like most genes in trypanosomatid protozoa(22) .
Additional posttranscriptional regulation of gp63 may lie in its posttranslational modifications. The N-terminal modifications of gp63 have been predicted from the gene sequences(13) . Aside from the signal peptide, a propeptide is presumably cleaved from the N-terminal end of the nascent gp63 by autocatalytic processing, thereby unblocking the active site via the ``cysteine switch'' mechanism(23, 24) . In matrixins(25) , this processing involves the HEXXH domain wherein the His and Glu residues are essential for the catalytic activity of these metalloprotease families in zinc binding and peptide hydrolysis, respectively(1) . Whether these residues of gp63 function analogously and are involved in the autocleavage of propeptide have not been examined in Leishmania cells.
The C-terminal
modification of gp63 presumably involves cleavage of a signal peptide
upon the addition of the glycosyl phosphatidylinositol (GPI) ()anchor(26) , for example, at Asn-577 of the Leishmania major sequence(27) . Regulation of gp63 at
this step has been suggested previously by transfecting Leishmania with Trypanosoma gene encoding GPI-phospholipase C (PLC),
which deprives gp63 of the GPI anchor, resulting in its extracellular
release(28) . In contrast, when mutated at their GPI addition
site, placental alkaline phosphatase (29) and decay
accelerating factor (30) both retain their hydrophobic
C-terminal extensions, forming homoaggregates susceptible to
intracellular degradation. Mutation of gp63 at the predicted GPI
addition site by the same approach is of interest not only for further
characterization of this site but also for elucidation of its
differences from the mammalian counterpart.
gp63 is also posttranslationally modified by N-glycosylation (8) . The functional significance of N-glycosylation was thought to stabilize gp63 and its enzymatic activity(31) , consistent with the observations of other glycosylated proteases, e.g. tissue plasminogen activator (32) and neutral endopeptidase(33) . Conservation of at least three potential sites of N-glycosylation in all gp63 gene sequences obtained so far (13, 14, 15, 16, 17, 18, 19) lends credence to this proposal, although work with chemically or enzymatically deglycosylated gp63 yielded inconsistent results(34) .
In the present study, posttranslational modifications of gp63 were examined by site-directed substitution of residues tentatively identified as important from gene sequences. Here, evidence is provided from transfection studies with the mutant genes for the functional significance of the residues in zinc binding/catalytic activity, N-glycosylation, and GPI addition. Evidence obtained suggests that these posttranslational modification pathways may be regulated to modulate the expression of gp63 as a functional protease at different stages in the life cycle of Leishmania.
Cloning and Site-directed Mutagenesis of gp63 Genes- Fig. 1shows the amino acid residues of gp63 substituted by oligonucleotide-directed mutagenesis (35) of the genes using a phagemid mutagenesis kit (Bio-Rad). Templates used were the gp63 gene from L. major(13) and in two cases from that of Leishmania amazonensis (GenBank accession no. L46798), both being cloned into a Leishmania-specific expression vector (pX-gp63)(36) . Antisense oligonucleotides used for mutagenesis contain 1-2 mismatched bases within the triplet codon of interest (see legend to Fig. 1for the oligonucleotide sequences used). Site-specific mutations were verified by sequencing selected clones using oligonucleotide primers prepared from the gene sequence about 100 base pairs downstream of the modified sites (see legend to Fig. 1for the primers used for sequencing). The resultant plasmids are designated on the basis of the amino acid residue mutated, its residue number, and respective substitution. Thus, E265D is a mutant in which Glu-265 is replaced with Asp in the pX-gp63 construct.
Figure 1: Functional residues predicted from gp63 gene sequences and their substitutions. The functional motifs and their residues predicted from gp63 gene sequences are indicated. The site-specific substitutions of these residues are shown below their respective positions. The residues subjected to mutagenesis and the corresponding oligonucleotides (boldface triplet codons with underlined base substitutions) used are: His-264, 5`-GCGCCATCTC-GTG/A or AACGTGACGACA-3`; His-268, 5`-AGCCGAGCGCGTG/A or TCGCCATCTCG-3`; Glu-265, 5`-CGTGCGCCATC/GTCTGCGTGACG-3`; Tyr-254, 5`-AGCTGGTCGTA/GTCCGCGACGCA-3`; Asn-300, 5`-CCGTGCTGCTG/TTT/GGATCACGGGA-3`; Asn-407, 5`-CCTCGCTCTCA/TTT/GGCAGAACATG-3`; Asn-534, 5`-CCGGCGTGCAG/TTT/GGGTGTAGTCG-3`; Asn-577, 5`-CAGCCGCCGTGT/AT/GGCCGCCGTCC-3`. Primers used for sequencing the above mutant genes to verify the site-specific mutations are: 5`-CCGTGCTGCTGTTGATCA-3` for mutations in the -HEXXH- or -SRYD- motifs, 5`-TGAGCTCGTCCTGCGCGT-3`, 5`-AGTAGTCCATGAAGGCGG-3`, 5`-TGCCCTGGCACACCTCCA-3`, and 5`-GGCGTGCGCCTGCTCCGCC-3` for mutants in the first, second, and third N-glycosylation and GPI addition sites, respectively.
To assess the release of gp63, promastigotes were labeled for 1 h at
a density of 10 cells/ml in Hanks' balanced salt
solution containing 50-100 µCi of
EXPRE
S
S Met/Cys mixture (DuPont NEN)
(specific activity =
1000 Ci/mmol). Cells were washed twice
in Medium 199 and resuspended in this medium containing 0.1% bovine
serum albumin (3
10
cells/ml). At different
intervals after incubation for up to 48 h, spent media were collected
and cleared by centrifugation at 50,000
g. Labeled
gp63 were immunoprecipitated from these supernatants for 1 h at 4
°C with 1/200 dilution of the anti-gp63 antiserum. Immune complexes
were collected by Protein A-Sepharose (Sigma), followed by washing and
autoradiography of these samples were performed as
described(10, 12) .
Figure 3: Western blot and gel-protease analyses of wild-type and mutant gp63 in Leishmania transfectants. Panel A, Coomassie Blue staining of SDS-PAGE gel showing comparable loading of all samples; Panel A`, Western blot with rabbit antiserum raised against p36, a constitutively expressed Leishmania NADPH-oxidoreductase; panel B, immunoblot with rabbit polyclonal sera raised against affinity-purified gp63, showing relative level of gp63 expression by wild-type and mutant genes in the transfectants; panel C, gelatin-containing gel for proteolytic activities of the same samples. Lane 1, parental gp63-deficient variant; lanes 2I and 2II, two independent transfectants with the wild-type gene; lanes 3-15, transfectants with the gp63 site-specific mutants loaded in the same order as those in Fig. 1and Table 1.
Figure 2:
Dot- and Northern blot analysis of the
total RNA from Leishmania transfected with wild-type and
mutant gp63 genes. Panel A, 2-fold serial dilutions of total
RNA probed with the gp63 gene. Panel B, duplicate blot of panel A probed with -tubulin gene of L. amazonensis to demonstrate equal sample loading. Lane 1,
transfectants with wild-type gp63; lanes 2 and 3,
transfectants with vector pX alone and non-transfected parental cells,
respectively; lanes 4-12, transfectants with mutant
genes, i.e. H264F, E265D, H268Y, Y254D, D577L, N300Q,
N300Q/N407Q, N407Q/N534Q, N300Q/N407Q/N534Q. Panel C, Northern
blot probed with the 2.2-kilobase pair gp63 gene. Lane 1,
transfectants with vector pX alone; lanes 2-8,
representative transfectants with the following genes: wild-type,
H264F, E265D, D577L, N300Q, N300Q/N407Q, and N300Q/N407Q/N534Q. RNA
samples were loaded at 10 µg/lane. See Fig. 1and
``Experimental Procedures'' for details of the mutants and
their preparations.
As reported previously(10, 12) , the
deficient variant used for transfection was found to contain a low
background of gp63 ( Table 1and Fig. 3, B and C, lane 1). The level of gp63 was substantially
increased by transfection of the variant with the wild-type gene (Table 1, Fig. 3, B and C, lanes 2I and 2II). Densitometry of their relative banding
intensity shows an average of 18-fold difference (Table 1,
Expression level; lanes 1 versus lanes 2I and 2II).
Comparison of the transfectants with and without the gp63 gene for
their surface caseinolytic activity revealed an even greater difference (Table 1, Caseinolytic activity; lanes 1 and 2I and 2II). It was estimated that 95% of the gp63 in
the transfectants is attributable to the expression of electroporated
genes. Coupled with the different electrophoretic mobility of their
products in the native gel, this level of the overexpression makes it
possible to assess the qualitative and quantitative effects of
mutagenesis on gp63. Unless specified, mutants referred to below were
produced with the gp63 gene of L. major.
Although the products of E265D are enzymatically inactive, they appear to have been appropriately processed posttranslationally, judging from their identity in electrophoretic mobility to the wild-type gp63 (Fig. 3B, lane 5 versus lane 2). This suggests that the cleavage of propeptide from the nascent gp63 does not require an ``intramolecular autocleavage,'' as found for the proenzyme activation in the matrixin family(25) . The proposal that gp63 may undergo a similar process of autocleavage (24) via the ``cysteine switch'' mechanism (23) remains possible, assuming that the propeptide is cleaved by an ``intermolecular'' processing event. In this instance, it may involve the action of either a different protease or the endogenous wild-type enzyme on the mutant. Additional work is needed to elucidate this important step in the regulation of gp63 maturation into a functional protease.
Figure 4: Extracellular exit of gp63 and characterization of the released gp63 from the transfectant with N577L, the GPI addition site mutant. Panel A, autoradiography of immunoprecipitated gp63 from culture supernatants of radiolabeled transfectants with either the wild-type gene (left panel) or the GPI mutant (N577L) (right panel). Spent medium was collected for immunoprecipitation with anti-gp63 antiserum at the time intervals as indicated. Panel B, immunoblot analysis of gp63 immunoprecipitated from supernatants of N577L transfectants (right two lanes) and from purified wild-type (Pur-) gp63 (left two lanes) with anti-gp63 (upper) and anti-CRD (lower) antisera. + and -, with or without GPI-PLC digestion.
The GPI addition site of gp63 differs in several aspects from that of other GPI-anchored membrane molecules. The abundant release of N577L mutant products indicates that the GPI anchor is the dominant, if not the only, mechanism for membrane anchoring of the gp63 in question and that the GPI addition site is quite specific. In contrast, other GPI-anchored molecules after similar mutations often become sequestered and degraded intracellularly(29, 30) , except in rare cases where an alternative GPI addition site may be used(45) . The results obtained from the N577L mutant raise the possibility that the addition of GPI to gp63 may regulate this molecule to act as a protease on the surface and/or after extracellular exit. This mode of regulation as a general phenomenon for GPI-anchored molecules deserves further study.
Clearly, the deglycosylated mutant products (Fig. 3, B and C, lanes 10-15) are less abundant than those from the wild-type gene or other mutant genes (lanes 2, 5, and 8). The low level of deglycosylated products is not due to their release, since only negligible amounts of gp63 were found in the medium of these transfectants (data not shown) as the wild-type transfectants (Fig. 4A, pX-gp63). Densitometry of the banding intensity further indicates that the levels of gp63 in single deglycosylation mutants (Table 1, lanes 10-12) and double or triple deglycosylation mutants are 2.5- and 13-fold less, respectively, than that of the fully glycosylated gp63 from the wild-type gene (Table 1, lanes 2I and 2II). The reduction is more profoundly affected by the number of glycosylation sites obliterated than their position in the molecule. It is noteworthy that the endogenous gp63 is also reduced in these transfectants, especially those with double or triple deglycosylation mutants (Fig. 3, B and C, lanes 13-15, solid arrow) (see ``Discussion''). The results thus suggest that the extent of N-glycosylation of gp63 affects its cellular level, presumably by stabilizing its structural integrity against proteolytic degradation(46) . The role of glycosylation is indeed thought to endow nascent peptides with proper local conformation for correct folding during cotranslation or transport into the RER(47) . N-Glycosylation of gp63 does not seem to be required for their expression on the cell surface, since deglycosylated products appear on the intact transfectants, as suggested by their increased caseinolytic activity (Table 1, lanes 10-15) and surface reactivity with anti-gp63 antibodies (data not shown).
All N-glycosylation mutant products, including those completely deglycosylated, are proteolytically active under the assay conditions used with either gelatin or azocasein ( Table 1and Fig. 3C, lanes 10-15) or fibrinogen (data not shown) as the substrate. Interestingly, semi-quantitative analysis of proteolytic activity by laser densitometry of zymograms suggests that deglycosylation may actually increase the specific gelatinolytic activity of gp63 (data not shown), although the total activity of deglycosylated gp63 appears lower than the wild-type protein (Fig. 3C and Table 1, Gelatinolysis, lanes 10-15 versus lane 2). Also, the surface caseinolytic activity of the transfectants with the deglycosylation mutants is only slightly lower, if at all, than that of the transfectants with the wild-type gene (Table 1, Caseinolysis, lanes 10-15 versus lane 2). These observations suggest that the specific caseinolytic activity of deglycosylated mutant products increases on the cell surface, since the amounts of these products decrease significantly with increasing level of deglycosylation. Our results are thus at variance with previous findings of no changes either in the quantity of gp63 or its specific proteolytic activities with enzymatically and chemically deglycosylated gp63(34) . Perhaps deglycosylation by non-genetic means is less complete, leading to the different observations reported. Glycosylation of other proteases may impart either positive or negative effects on their enzymatic activities. For example, the cleavage of plasminogen by tissue plasminogen activator increases by 2-4-fold in the absence of one of its three N-linked glycans(32) , while the activity of neutral endopeptidase decreases after deleting three of its five N-glycans(33) . In both cases, the differences observed are thought to result from changes in intrinsic catalytic activity, but not substrate binding. To address this issue in the case of gp63, it is necessary to purify genetically deglycosylated products for comparison with wild-type proteins to determine their kinetics of enzyme activity against specific substrates.
Here, we examined regulatory functions of posttranslational modifications on the Leishmania HEXXH metalloprotease. Insofar as is known, it is the only family of these proteases so extensively modified posttranslationally by both N-glycosylation and GPI addition in addition to the N-terminal end processing. While a number of metalloproteases are known to be heavily glycosylated, few are GPI-anchored, i.e. human carboxypeptidase M and yeast vacuolar dipeptidase, but neither contains HEXXH (see (1) ). Additional significance more specific to this group of organisms includes their apparent absence of transcriptional regulation of gene expression (22) and the implication of gp63 in the infectivity of these parasites(6) . Thus, posttranslational modifications represent potentially important steps in regulating the expression of this and possibly other similar molecules.
Several favorable factors made our approach to study posttranslational modifications of gp63 by site-specific mutagenesis possible. 1) The gp63 genes are highly conserved and functional residues in the putative motifs already identified(13, 14, 15, 16, 17, 18, 19) for mutagenesis (see Fig. 1); 2) the use of Leishmania-specific vector pX (36) for the transfections allowed consistent transcription of the electroporated genes (Fig. 2); 3) the gp63-deficient variant (10) used provides a suitable recipient host in the absence of Leishmania gp63 null mutants(20, 48) ; 4) electroporated gene products are sufficiently abundant for the specific immunologic and protease assays used to achieve adequate quantitative and qualitative comparisons ( Fig. 3and Table 1). The results obtained here with the posttranslational regulation of gp63 include several observations that have not been reported previously with other glycosylated HEXXH metalloproteases or GPI-anchored molecules. Specifically, gp63 appears not to require an intramolecular autolytic mechanism for cleaving its N-terminal propeptide, requires zinc binding and N-glycosylation to maintain its intracellular integrity, and is released with the loss of its GPI anchor. How these events, presumably all initiated in the RER, are coordinated to regulate gp63 are not known, but several observations pose interesting questions that are worthy of further consideration.
Interestingly, substitution of either His residue in HEXXH leads to no detectable mutant gp63 in the transfectants with all four independently prepared mutants ( Table 1and Fig. 3, B and C, lanes 3, 4 and 6, 7), while it is only reduced in transfectants with all the deglycosylation mutants (lanes 10-15, open arrow). Most unexpected is the observation that the endogenous gp63 is simultaneously reduced, but only in the transfectants with deglycosylation mutants (Fig. 3, B and C, lanes 10-15, solid arrow). This disparity seen between the two groups of mutants may signify functional differences of zinc binding and N-glycosylation in their respective contributions to the intracellular stability of gp63. Mutations of the His residues in HEXXH presumably deprives gp63 of a zinc atom. Such mutations may simply change the conformation of gp63 more drastically than deglycosylation, thereby rendering His mutants more susceptible than the deglycosylation mutants to intracellular degradation. However, this scenario does not accommodate the simultaneous reduction of the endogenous gp63 seen only in the transfectants with deglycosylation mutants. One possible explanation may lie in the fact that gp63 is a homopolymer in its native state(3, 8) . If monomeric deglycosylated mutant products and endogenous fully glycosylated gp63 in the transfectants co-polymerize randomly, it is possible to envision the formation of mutant/wild-type heteropolymers, leading to their intermediate susceptibility to intracellular degradation and thus a partial reduction of both wild-type and mutant gp63 seen (Fig. 3, B and C, lanes 10-15). Conformational changes of the His mutants may be so drastic that they become too short-lived or too deformed to co-polymerize with the endogenous gp63, thereby leaving the latter undisturbed (Fig. 3B, lanes 3, 4, 6, and 7, solid arrow).
It appears that both the N and C termini of gp63 are processed differently from other proteases or GPI-anchored proteins. Unlike the zymogens of many secretory proteases, e.g. matrixins(25) , released gp63 is proteolytically active and approaches the size of the mature protein. Thus, the predicted N-terminal 59-amino acid propeptide of the products must have been already cleaved intracellularly before their release and by a mechanism other than autoendopeptidolysis. The complete extracellular exit of N577L mutant products is an observation that has not been reported previously with other GPI-anchored molecules. In other GPI-anchored molecules studied so far, the addition of GPI is accompanied with the cleavage of their hydrophobic C-terminal end for exit from the RER for ectocellular targeting(29, 30) . Our GPI-less mutant products appear to exit not only from the RER but also from the cells. Conceivably, like wild-type gp63 or other membrane proteins, the mutant products may be transported in vesicles along the normal route for surface expression. Upon fusion of these vesicles with the plasma membrane, however, the mutant products therein are set free because they lack a GPI anchor. This transport mechanism of the gp63 mutants deserves further study, especially with respect to the cleavage of their C-terminal end and their insusceptibility to degradation, in contrast to other GPI-anchored molecules.
The work presented here underscores the potential importance of multiple posttranslational modifications of gp63 in the regulation of its expression. It is conceivable that the extreme conditions encountered by Leishmania during their life cycle in both insect and mammalian hosts may up- or down-regulate their N-glycosylation, GPI addition, and terminal end processing and, hence, the expression of gp63. Indeed, up-regulation of gp63 has been reported to play a role in Leishmania infection(5, 6, 10, 21) .