Serine-Type Carboxypeptidase KexA of Aspergillus oryzae Has Broader Substrate Specificity than Saccharomyces cerevisiae Kex1 and Is Required for Normal Hyphal Growth and Conidiation

Abstract

Aspergillus oryzae has an ortholog of Saccharomyces cerevisiae KEX1, termed kexA. A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth of A. oryzae and that it might be involved in hyphal branching.

TEXT

Saccharomyces cerevisiae Kex1 is one of the processing enzymes that have a transmembrane domain in the C-terminal region and localize in the Golgi membrane (7). The enzyme has an active serine residue at its active site and functions as an exopeptidase, displaying specificity for proteins and peptides with C-terminal basic amino acid residues (6, 8, 13). Kex1 is involved in the proteolytic processing of α-factor and of S. cerevisiae killer toxin (3, 4, 5, 8, 12, 17). In addition, recent studies have shown that Kex1 plays a more general role in yeast (9, 10). A whole-genome BLAST search revealed that the KEX1 orthologous gene is also present in the genome of the Aspergillus species; however, there are no reports on the enzymatic or functional properties of the Kex1 ortholog in the filamentous fungi. An ortholog of KEX1 identified in Aspergillus oryzae, termed kexA (Database of Genomes Analyzed at NITE accession no. AO090005001632; National Center for Biotechnology Information gene ID 5990481), was predicted to encode a 625-amino-acid protein which shares 38% identity with Kex1.

To investigate the enzymatic properties of KexA, we attempted purification of a truncated form of KexA (amino acid residues 1 to 526 of KexA), which lacked the potential transmembrane region and the succeeding C-terminal sequence (amino acid residues 527 to 625 of KexA), by using a protein expression system with the pIECS3 vector and A. nidulans FGSC A89 (15) (Fig. 1A). A protein of approximately 60 kDa, which was consistent with the molecular mass calculated from the deduced amino acid sequence of the truncated KexA (58,821 Da), was found in an intracellular fraction extracted from the cultured hyphae of the truncated-KexA-overproducing strain (Fig. 1B), and the fraction displayed higher peptidase activity for Bz-Gly-Arg than that of the host strain (data not shown). Thus, the truncated KexA was purified from the fraction by using anion-exchange (HiTrap QXL and Resource Q [GE Healthcare, Buckinghamshire, United Kingdom]) and gel filtration (Sephacryl S200 [GE Healthcare]) columns. The purified protein appeared as two bands by SDS-PAGE (Fig. 1C); however, peptide mass fingerprinting analysis showed that both bands corresponded to a protein encoded by kexA. Therefore, the protein was used for enzymatic characterization as the purified recombinant truncated KexA. Those bands were also found in the crude intracellular fraction of the truncated-KexA-overproducing strain (Fig. 1B), suggesting that the overproduced truncated KexA could be somewhat degraded by a protease(s) from the host strain in the cell. But the protease(s) responsible for degradation of the truncated KexA was removed by the purification steps, as even after the purified truncated-KexA fraction was incubated overnight at 30°C, degradation of the purified protein was not observed.

Fig 1.

Plasmid used for construction of the truncated-KexA-overproducing strain (A) and SDS-PAGE profiles of the intracellular fractions from the host strain and the truncated-KexA-overproducing strain (B) or the purified truncated-KexA (C). (A) The truncated kexA (bp 1 to 1723 of kexA and a stop codon) gene encoded a protein corresponding to residues 1 to 526 of KexA. It was amplified by PCR using the genomic DNA obtained from A. oryzae RIB40 as the template DNA. aurA is an aureobasidin A resistance gene from A. nidulans. (B) SDS-PAGE was performed using the intracellular fractions from the host strain (lane 1) and the truncated-KexA-overproducing strain (lane 2), and separated proteins were stained with Coomassie brilliant blue. The intracellular fractions were prepared as follows. Mycelia cultured in the induction medium (5% soluble starch [wt/vol], 1% yeast extract [wt/vol], 2% Bacto peptone [wt/vol], 0.5% KH₂PO₄ [wt/vol], 0.5% MgSO₄ [wt/vol], 1% rice bran [wt/vol], 0.5 mM arginine; pH 3.5) at 30°C for 3 days were collected by filtration and ground with liquid nitrogen. Subsequently, 10 ml of cold 20 mM phosphate buffer (pH 7.0) was added to the ground mycelia, and the cell suspension was centrifuged at 17,000 × g for 30 min at 4°C. The obtained supernatants were used as samples for SDS-PAGE. (C) The purified truncated KexA was visualized as two bands by SDS-PAGE (arrowheads). Both bands were identified as a translation product of kexA by peptide mass fingerprinting.

The truncated KexA was treated with peptide N-glycosidase F; however, the treated protein exhibited no shift in mobility on SDS-PAGE gel (data not shown). The result suggested that the truncated KexA would have no N-linked carbohydrate chains. The truncated KexA released amino acid residues from the C terminus of angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) in a sequential manner (data not shown), indicating that the truncated KexA has a carboxypeptidase activity. Similar to Kex1, the truncated KexA also hydrolyzed substrates containing a C-terminal basic amino acid residue, with a preference for arginine over lysine as the C-terminal amino acid (Table 1), and showed the highest activity around pH 6.0 (Table 2). However, although Kex1 hydrolyzed only substrates with C-terminal basic amino acid residues (6), the truncated KexA exhibited carboxypeptidase activity for the peptides with a C-terminal leucine residue, such as Z-Tyr-Leu, Z-Phe-Leu, Z-Phe-Tyr-Leu, and angiotensin I. Kex1 possesses a highly acidic region ahead of the transmembrane region. A previous study suggested that the region interferes with access of the substrate to the active site of Kex1 (14). In contrast to Kex1, there is no highly acidic region in KexA or truncated KexA. Thus, the absence of this region in truncated KexA may be one of the possible causes of the somewhat broader substrate specificity of the protein compared with that of Kex1. The pH and thermal stability of the truncated KexA and effect of protease inhibitors on the activity are shown in Table 2.

Table 1.

Substrate specificity of the truncated KexA^a

Substrate	Sp act (mkat/kg)
Z-Leu-Tyr	ND
Z-Tyr-Leu	1.2
Z-Phe-Leu	1.2
Z-Phe-Tyr-Leu	38.9
Z-Glu-Tyr	ND
Z-Gly-Pro-Leu-Gly	tr
Z-Gln-Gly	ND
Z-Val-Gly	ND
Z-Ala-Glu	tr
Bz-Gly-Arg	3.0
Z-Gly-Lys	1.1
Z-Gly-Phe	ND
Z-Gly-Leu	ND
Z-Gly-Pro	ND

^aThe substrate specificity was measured at pH 5.5 by the ninhydrin assay, as described previously (15). Z, benzyloxycarbonyl; Bz, benzoyl; ND, not detectable.

Table 2.

Enzymatic properties of the truncated KexA^a

Property or measurement	Value
Optimum pH	5.5
pH stabilityb	5–8
Thermal stability (°C)c	40
Effect on KexA activity (%) of protease inhibitord
PMSF	32
E-64	92
MIA	10
Pepstatin A	99
EDTA	86

^aThe carboxypeptidase activity was measured by ninhydrin assay as described previously (11, 16) using 1 mM Bz-Gly-Arg dissolved in 50 mM phosphate buffer (pH 5.5) as the substrate.

^bpH range in which the enzyme had residual activity of over 60% relative to maximum activity.

^cMaximum temperature at which the enzyme had residual activity of over 60% relative to maximum activity.

^dValues are relative activities and were quantified as the percent activity of each reference that contained water or dimethyl sulfoxide instead of inhibitor. PMSF, phenylmethylsulfonyl fluoride; E-64, N-[N-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]-agmatine; MIA, monoiodo acetic acid.

kexA was constitutively expressed in A. oryzae grown on either liquid medium or agar plates (see Fig. S1 in the supplemental material), implying that KexA may be involved in growth of the fungus. Therefore, a kexA deletion strain was generated in order to verify the functionality of KexA in A. oryzae (see Fig. S2 in the supplemental material). When the ΔkexA strain was inoculated on both CDEM (Czapek-Dox minimal medium supplemented with 30 μg/ml methionine and 70 mM monosodium glutamate instead of sodium nitrate) and potato dextrose agar plates, the radial growth speeds of the strain were slightly lower than those of the host strain (Fig. 2A and B), and the frequency of hyphal branching of the strain was lower than that of the host strain on CDEM slide culture (Fig. 2C). The low frequency of hyphal branching of the ΔkexA strain implied that the strain possessed defects in the cell wall. However, the growth of the ΔkexA strain was scarcely affected by cell wall-interfering reagents, including Calcofluor white and Congo red, and the glucan synthase inhibitor micafungin, suggesting that KexA might not participate in the major cell wall construction of the hyphae (data not shown). In addition to those phenotypes, the ΔkexA strain appeared to form fewer conidia than the host strain on the agar plates (Fig. 2A and B). In the host strain, transcription of brlA, one of the essential genes for conidium formation (1, 2), was detected in 48-h-cultured hyphae on potato dextrose agar plate, and it increased in a time-dependent manner (Fig. 3). However, brlA was not transcribed in the ΔkexA strain cultured for 48 or 72 h. At 96 h of culture, the transcriptional level of brlA in the ΔkexA strain reached a level similar to that observed in the host strain cultured for 72 h, indicating that transcription of brlA in the ΔkexA strain was delayed compared with that in the host strain (Fig. 3). Notably, the transcription of brlA was delayed but not suppressed completely. This effect suggested that kexA was required for normal hyphal growth of A. oryzae, and the delay of growth of the ΔkexA strain—resulting from loss of KexA—gave rise to a transcriptional lag of brlA, which would cause a delay in conidium formation. We also constructed a kexA-overexpressing strain and observed its phenotypes (see Fig. S2 in the supplemental material). As shown in Fig. 2D, the radial growth speed of the kexA-overexpressing strain was lower than that of the control strain. In addition, the kexA-overexpressing strain revealed a hyperbranching phenotype on CDMAL (Czapek-Dox minimal medium supplemented with 2% maltose) slide culture (Fig. 2E). The ΔkexA strain exhibited scarcely branched hyphae, whereas the kexA-overexpressing strain exhibited a hyperbranching phenotype. Thus, we consider that KexA may be involved in hyphal branching through proteolytic processing of the protein(s) necessary for hyphal branching and/or its regulatory factor(s).

Fig 2.

Phenotypes of the host strain, the ΔkexA strain, the control strain, and the kexA-overexpressing strain. A total of 3 μl of 2 × 10⁶ conidia/ml from the host strain and the ΔkexA strain were spotted onto CDEM (A) and potato dextrose (B) agar plates and cultured at 30°C. (C) Hyphae and hyphal tips of the host strain and the ΔkexA strain. Conidia were inoculated on slides coated with a thin layer of CDEM agar medium and incubated at 30°C for 2 days. (D) A total of 3 μl of 2 × 10⁶ conidia/ml from the control strain, which was constructed by transformation using an empty plasmid, and the kexA-overexpressing strain was spotted onto a CDMAL (Czapek-Dox minimal medium supplemented with 2% maltose as the sole carbon source for overexpression) agar plate and cultured at 30°C. (E) Hyphae and hyphal tips of the control strain and the kexA-overexpressing strain. Conidia were inoculated onto slides coated with a thin layer of CDMAL agar medium and incubated at 30°C for 2 days.

Fig 3.

Semiquantitative RT-PCR analysis of kexA and brlA in the host strain (H) and the ΔkexA strain (Δ). mRNAs were prepared from the hyphae cultured on potato dextrose agar plates at 30°C for 48, 72, and 96 h. An oligo(dT) primer was used for reverse transcription, and the primer pairs kexA-RT-F and kexA-RT-R (see Fig. S1 in the supplemental material) and brlA-RT-F (5′-ACGGTTGAAGTCGACTGCCACTC-3′) and brlA-RT-R (5′-CCATCAACACTGTATTCGCGGCT-3′) were used to amplify kexA and brlA, respectively. The gene encoding γ-actin was amplified for the quantitative control of cDNAs by using the primers actin-F and actin-R (see Fig. S1). PCR was performed for 28 cycles using a step-cycle program set at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. A negative-control PCR analysis using each mRNA as a template showed no amplified fragments (data not shown).