Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation.

Herpesviruses encode an essential serine proteinase called assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of assemblin mutants. I-site cleavage of the mutated assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV assemblin). These results indicate that assemblin is able to form active multimeric structures that may be of functional importance.     

Cytomegalovirus assemblin (pUL80a): cleavage at internal site not essential for virus growth; proteinase absent from virions

The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called assemblin. I-site cleavage cuts assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of assemblin nor the presence of assemblin in the mature virion.     

Cleavage of human cytomegalovirus protease pUL80a at internal and cryptic sites is not essential but enhances infectivity

The cytomegalovirus (CMV) maturational protease, assemblin, contains an "internal" (I) cleavage site absent from its homologs in other herpesviruses. Blocking this site for cleavage did not prevent replication of the resulting I(-) mutant virus. However, cells infected with the I(-) virus showed increased amounts of a fragment produced by cleavage at the nearby "cryptic" (C) site, suggesting that its replication may bypass the I-site block by using the C site as an alternate cleavage pathway. To test this and further examine the biological importance of these cleavages, we constructed two additional virus mutants-one blocked for C-site cleavage and another blocked for both I- and C-site cleavage. Infectivity comparisons with the parental wild-type virus showed that the I(-) mutant was the least affected for virus production, whereas infectivity of the C(-) mutant was reduced by approximately 40% and when both sites were blocked virus infectivity was reduced by nearly 90%, providing the first evidence that these cleavages have biological significance. We also present and discuss evidence suggesting that I-site cleavage destabilizes assemblin and its fragments, whereas C-site cleavage does not.     

Cytomegalovirus assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells.

The cytomegalovirus (CMV) serine proteinase assemblin is synthesized as a precursor that undergoes three principal autoproteolytic cleavages. Two of these are common to the assemblin homologs of all herpes group viruses: one at the maturational site near the carboxyl end of the precursor and another at the release site near the midpoint of the precursor. Release-site cleavage frees the proteolytic amino domain, assemblin, from the nonproteolytic carboxyl domain of the precursor. In CMV, a third autoproteolytic cleavage at an internal site divides assemblin into an amino subunit (An) and a carboxyl subunit (Ac) of approximately the same size that remain associated as an active "two-chain" enzyme. We have cloned the sequences encoding An and Ac as separate genes and expressed them by transfecting human cells with recombinant plasmids and by infecting insect cells with recombinant baculoviruses. When An and Ac from either simian CMV or human CMV were coexpressed in human or insect cells, active two-chain assemblin was formed. This finding demonstrates that An and Ac do not require synthesis as single-chain assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the independently expressed An and Ac subunits was demonstrated by coimmunoprecipitation experiments, and efforts to disrupt the complex indicate that the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain assemblin formed from independently expressed An and Ac also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain assemblin, (ii) R-site cleavage is not essential for the activity of two-chain recombinant assemblin, and (iii) the human CMV and simian CMV An and Ac recombinant subunits are functionally interchangeable.     

Cytomegalovirus capsid protease: biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin)

We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na(2)SO(4) reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.     

Enzymatic activities of human cytomegalovirus maturational protease assemblin and its precursor (pPR, pUL80a) are comparable: [corrected] maximal activity of pPR requires self-interaction through its scaffolding domain

Herpesviruses encode an essential, maturational serine protease whose catalytic domain, assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered "tail" (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.     

The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans

The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn²⁺, Cd²⁺, and Co²⁺, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707–4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans.     

Activation mechanism of pro-astacin: role of the pro-peptide,tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing

Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family.     

Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site.     

Changing the protease specificity for activation of a flavivirus, tick-borne encephalitis virus

The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.     

Probing the catalytically essential residues of the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus

The alpha-L-arabinofuranosidase D3 from Thermobacillus xylanilyticus is an arabinoxylan-debranching enzyme which belongs to family 51 of the glycosyl hydrolase classification. Previous studies have indicated that members of this family are retaining enzymes and may form part of the 4/7 superfamily of glycosyl hydrolases. To investigate the active site of alpha-L-arabinofuranosidase D3, we have used sequence alignment, site-directed mutagenesis and kinetic analyses. Likewise, we have shown that Glu(28), Glu(176) and Glu(298) are important for catalytic activity. Kinetic data obtained for the mutant Glu(176)-->Gln, combined with the results of chemical rescue using the mutant Glu(176)-->Ala, have shown that Glu(176) is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu(176)-->Ala, indicated that alpha-L-arabinofuranosidase D3 hydrolyses glycosidic bonds with retention of the anomeric configuration. The results of similar chemical rescue studies using other mutant enzymes suggest that Glu(298) might be the catalytic nucleophile and that Glu(28) is a third member of a catalytic triad which may be responsible for modulating the ionization state of the acid-base and implicated in substrate fixation. Overall, these findings support the hypothesis that alpha-L-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.     

Identification of active-site amino acid residues in the Chiba virus 3C-like protease

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.     

Mutation of the f-protein cleavage site of avian paramyxovirus type 7 results in furin cleavage, fusion promotion, and increased replication in vitro but not increased replication, tissue tropism, or virulence in chickens

We constructed a reverse genetics system for avian paramyxovirus serotype 7 (APMV-7) to investigate the role of the fusion F glycoprotein in tissue tropism and virulence. The AMPV-7 F protein has a single basic residue arginine (R) at position -1 in the F cleavage site sequence and also is unusual in having alanine at position +2 (LPSSR↓FA) (underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.). APMV-7 does not form syncytia or plaques in cell culture, but its replication in vitro does not depend on, and is not increased by, added protease. Two mutants were successfully recovered in which the cleavage site was modified to mimic sites that are found in virulent Newcastle disease virus isolates and to contain 4 or 5 basic residues as well as isoleucine in the +2 position: (RRQKR↓FI) or (RRKKR↓FI), named Fcs-4B or Fcs-5B, respectively. In cell culture, one of the mutants, Fcs-5B, formed protease-independent syncytia and grew to 10-fold-higher titers compared to the parent and Fcs-4B viruses. This indicated the importance of the single additional basic residue (K) at position -3. Syncytium formation and virus yield of the Fcs-5B virus was impaired by the furin inhibitor decanoyl-RVKR-CMK, whereas parental APMV-7 was not affected. APMV-7 is avirulent in chickens and is limited in tropism to the upper respiratory tract of 1-day-old and 2-week-old chickens, and these characteristics were unchanged for the two mutant viruses. Thus, the acquisition of furin cleavability by APMV-7 resulted in syncytium formation and increased virus yield in vitro but did not alter virus yield, tropism, or virulence in chickens.     

Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate. The pH dependence of V and V/K for the E. coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism. To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256. Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in V(max), and 336- and 63-fold increases in the K(m) value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties. The V(max) for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively. The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume. The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction. The rescue efficiencies for the K176A mutant by primary amines were independent of the pK(a) value of the rescue agents (Bronsted coefficient, alpha = -0.004 +/-0.008). Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (k(cat)/K(m) = 2x10(6) M(-1) s(-1) and (b) the small primary deuterium kinetic isotope effects, (D)V = 1.3 and (D)V/K = 1.5, observed for the wild-type enzyme. Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant ((D)V = 3.0, (D)V/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding. The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity. These results indicate that Lys176 and Glu256 of the E. coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.     

X-ray snapshots of peptide processing in mutants of tricorn-interacting factor F1 from Thermoplasma acidophilum

The tricorn-interacting factor F1 of the archaeon Thermoplasma acidophilum cleaves small hydrophobic peptide products of the proteasome and tricorn protease. F1 mutants of the active site residues that are involved in substrate recognition and catalysis displayed distinct activity patterns toward fluorogenic test substrates. Crystal structures of the mutant proteins complexed with peptides Phe-Leu, Pro-Pro, or Pro-Leu-Gly-Gly showed interaction of glutamates 213 and 245 with the N termini of the peptides and defined the S1 and S1' sites and the role of the catalytic residues. Evidence was found for processive peptide cleavage in the N-to-C direction, whereby the P1' product is translocated into the S1 site. A functional interaction of F1 with the tricorn protease was observed with the inactive F1 mutant G37A. Moreover, small angle x-ray scattering measurements for tricorn and inhibited F1 have been interpreted as formation of transient and substrate-induced complexes.     

The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair

The conserved, structure-specific flap endonuclease FEN1 cleaves 5' DNA flaps that arise during replication or repair. To address in vivo mechanisms of flap cleavage, we developed a screen for human FEN1 mutants that are toxic when expressed in yeast. Two targets were revealed: the flexible loop domain and the catalytic site. Toxic mutants caused G(2) arrest and cell death and were unable to repair methyl methanesulfonate lesions. All the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5' flaps, the loop mutants exhibited partial ability to cut 5' flaps when an adjacent single nucleotide 3' flap was present. We suggest that the flexible loop is important for efficient cleavage through positioning the 5' flap and the catalytic site.     

Herpesvirus proteinase: site-directed mutagenesis used to study maturational, release, and inactivation cleavage sites of precursor and to identify a possible catalytic site serine and histidine.

The cytomegalovirus maturational proteinase is synthesized as a precursor that undergoes at least three processing cleavages. Two of these were predicted to be at highly conserved consensus sequences--one near the carboxyl end of the precursor, called the maturational (M) site, and the other near the middle of the precursor, called the release (R) site. A third less-well-conserved cleavage site, called the inactivation (I) site, was also identified near the middle of the human cytomegalovirus 28-kDa assemblin homolog. We have used site-directed mutagenesis to verify all three predicted sequences in the simian cytomegalovirus proteinase, and have shown that the proteinase precursor is active without cleavage at these sites. We have also shown that the P4 tyrosine and the P2 lysine of the R site were more sensitive to substitution than the other R- and M-site residues tested: substitution of alanine for P4 tyrosine at the R site severely reduced cleavage at that site but not at the M site, and substitution of asparagine for lysine at P2 of the R site reduced M-site cleavage and nearly eliminated I-site cleavage but had little effect on R-site cleavage. With the exception of P1' serine, all R-site mutations hindered I-site cleavage, suggesting a role for the carboxyl end of assemblin in I-site cleavage. Pulse-chase radiolabeling and site-directed mutagenesis indicated that assemblin is metabolically unstable and is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, the enzymatic amino half of the proteinase precursor. Among those tested, only 2 amino acids were identified as essential for activity: the single absolutely conserved serine and one of the two absolutely conserved histidines. When the highly conserved glutamic acid (Glu22) was substituted, the proteinase was able to cleave at the M and I sites but not at the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., protein conformation) role for this residue in determining substrate specificity.     

Shedding of TRAP by a Rhomboid Protease from the Malaria Sporozoite Surface Is Essential for Gliding Motility and Sporozoite Infectivity

Plasmodium sporozoites, the infective stage of the malaria parasite, move by gliding motility, a unique form of locomotion required for tissue migration and host cell invasion. TRAP, a transmembrane protein with extracellular adhesive domains and a cytoplasmic tail linked to the actomyosin motor, is central to this process. Forward movement is achieved when TRAP, bound to matrix or host cell receptors, is translocated posteriorly. It has been hypothesized that these adhesive interactions must ultimately be disengaged for continuous forward movement to occur. TRAP has a canonical rhomboid-cleavage site within its transmembrane domain and mutations were introduced into this sequence to elucidate the function of TRAP cleavage and determine the nature of the responsible protease. Rhomboid cleavage site mutants were defective in TRAP shedding and displayed slow, staccato motility and reduced infectivity. Moreover, they had a more dramatic reduction in infectivity after intradermal inoculation compared to intravenous inoculation, suggesting that robust gliding is critical for dermal exit. The intermediate phenotype of the rhomboid cleavage site mutants suggested residual, albeit inefficient cleavage by another protease. We therefore generated a mutant in which both the rhomboid-cleavage site and the alternate cleavage site were altered. This mutant was non-motile and non-infectious, demonstrating that TRAP removal from the sporozoite surface functions to break adhesive connections between the parasite and extracellular matrix or host cell receptors, which in turn is essential for motility and invasion.