Sequence Information

Proteases

Dipeptidyl peptidase - dppV

One research group has cloned the dppV gene (L48074), 3349 bp from strain CBS 144-89 (Beauvais et al., 1997).

The dppV gene codes for a dipeptidyl peptidase (O13479). The ORF encodes 721 amino acids (Predicted MW 79 000 Da Experimental MW 87-88 000 Da) and dppV contains seven introns of between 53 and 94 bp. The protein has an 18 amino acid signal peptide. The C-terminal end of DPPV has significant identity with the C-terminal 200 amino acids of class IV dipeptidyl peptidases from human, mouse and rat and to dipeptidyl aminopeptidase from S. cerevisiae. The DPPV protein also contains a putative catalytic triad (Ser-Asp-His) found in class IV dipeptidyl peptidases. However, DPPV does not release dipeptides diagnostic of any dipeptidyl peptidase class and DPPV has therefore been classed as a novel group V dipeptidyl peptidase. Deglycosylation of recombinant DPPV reduced its MW from 87-88 000 Da to 79 000 Da indicating that the disparity between theoretical and experimental MW resulted from glycosylation of the DPPV protein.

Paper:

Beauvais A, Monod M, Svab J, Kobayashi H, Diaquin M, Hovanessian A.G, Latge J.P.
"Biochemical and antigenic characterization of a new dipeptidyl-peptidase isolated from Aspergillu fumigatus."
J. Biol. Chem. 272: 0-0. 1997.

A novel dipeptidyl-peptidase (DPP V) was purified from the culture medium of Aspergillus fumigatus. This is the first report of a secreted dipeptidyl-peptidase. The enzyme had a molecular mass of 88 kDa and contained approximately 9 kDa of N-linked carbohydrate. The expression and secretion of dipeptidyl-peptidase varied with the growth conditions; maximal intra- and extracellular levels were detected when the culture medium contained only proteins or protein hydrolysates in the absence of sugars. The gene of DPP V was cloned and showed significant sequence homology to other eukaryotic dipeptidyl-peptidase genes. Unlike the other dipeptidyl-peptidases, which are all intracellular, DPP V contained a signal peptide. Like the genes of other dipeptidyl-peptidases, that of DPP V displayed the consensus sequences of the catalytic site of the nonclassical serine proteases. The biochemical properties of native and recombinant DPP V obtained in Pichia pastoris were unique and were characterized by a substrate specificity limited to the hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at a neutral pH optimum. In addition, we showed that DPP V is identical to one of the two major antigens used for the diagnosis of aspergillosis.

 

Dipeptidyl peptidase - dppIV

One research group has cloned the dppIV gene (U87950), 2352 bp from strain CBS 144-89 (Beauvais et al., 1997).

The dppIV gene encodes a class IV dipeptidyl peptidase (O14425). The ORF of dppIV encodes 767 amino acids (Predicted MW 85 816 Da, Experimental MW 95-100 000 Da) and the gene contains 1 intron of 54bp. The protein has an 14 amino acid signal peptide. The C-terminal 200 residues of DPPIV have significant identity with the C-terminal 200 residues from rat, mouse and human class IV dipeptidyl peptidases and with S. cerevisiae dipeptidyl aminopeptidase. The C-terminal 200 residues of DPPIV also contain a putative catalytic triad (Ser, Asp, His) found in class IV dipeptidyl peptidases and the serine protease consensus Gly-X-Ser-X-Gly. The DPPIV protein releases X-Pro and X-Ala peptides and is therefore classified as a class IV dipeptidyl peptidase. Recombinant DPPIV migrated at 95-100 000 Da, but after deglycosylation migrated as a discrete 85 000 Da protein indicating that the disparity between theoretical and experimental MW resulted from glycosylation of DPPIV.

Paper:

Beauvais A, Monod M, Wyniger J, Debeaupuis J.P, Grouzmann E, Brakch N, Svab J, Hovanessian A.G, Latge J.P.
"Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans."
Infect. Immun. 65(8): 3042-3047. 1997

Dpp's are recognized as playing an essential role in human physiology, these enzymes belong to different classes depending on the nature of the dipeptide released as well as their cellular localization. DppIV CD26 is a surface differentiation marker involved in the transduction of mitogenic signals in thymocytes and T lymphocytes (human, rat, mouse) and in cell matrix adhesion through specific inmteractions with fibronectin and collagen. DppIV encodes a secreted enzyme with a cleavable signal peptide, the enzyme has an apparent molecular weight of 95kDa and contains approximatley 10kDa of N-linked carbohydrate. The hydropathy profile of the deduced amino acid sequence of the Afu dppIV showed a highly hydrophobic amino-terminal end corresponding to the signal peptide.

 

Metalloprotease - mep20

One research group has cloned the mep20 gene (U24146), 1354 bp from strain isolate 13 (Ramesh et al., 1995).

The mep20 gene codes for a putative metalloprotease (Q09016). The ORF is predicted to encode 365 amino acids (Predicted MW 39 030 Da) and contains one intron of 59 bp. The A. fumigatus mep20 gene was isolated from a genomic library using a 282 bp fragment of the A. flavus mep20 gene as a probe. The A. fumigatus MEP20 protein has 68 % identity with the A. flavus MEP20 protein and the function of the A. fumigatus protein has been deduced because of its similarity to the A. flavus metalloprotease. The putative catalytic sequence His-Gly-Phe-Thr-His-Ala is found in the A. fumigatus MEP20 amino acid sequence at residue numbers 298 to 103. Data from the A. flavus MEP20 protein suggests that the A. fumigatus protein is processed to produce a 193 amino acid mature protein.

Paper:

Ramesh M.V, Sirakova T.D, Kolattukudy P.E.
"Cloning and characterisation of the cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus. "
Gene 165(1): 121-125. 1995

Ramesh and collegues isolated cDNA using degenerate primers based on the amino acid sequence of A. oryzae mep. First they cloned the A.flavus mep20 sequence, the Afu mep20 gene was isolated by screening Afu lambdaGEM12 genomic library, using the 282bp PCR fragment of Afl mep20 as a probe. A 1.4kb EcorI fragment from a hybridising clone was subcloned in pUC18 plasmid and sequenced. The presence of the intron was tested in five strains by PCR analysis, the results show that the intron contains the 5' splice donor (GTRNGT), 3' splice acceptor (YAG) and internal element (RCTRAC).

 

Metalloprotease - mep (Aspf5)

Two research group has cloned the mep gene:
( Z30424) 3075 bp from strain delta 18 ( Jaton-Ogay et al., 1994) and
( U24146), 1354 bp from strain isolate 13 ( Ramesh et al., 1995).

Table 1. below gives details of the sequence differences between L29566 and Z30424 (from the start codon to the stop codon only)

Table 1.

Position (bp of L29566)
L29566
Z30424
Amino acid position
Amino acid change
 
456
GGG
GGC
3
No change
 
471
GGA
GGG
 8
No change
 
537
CGG
CGT
30
No change
 
803
A
G
Intron
 
 
972
-
C
Intron
 
 
902
ACG
ACC
135
No change
 
906
AAG
CAG
137
K(L29566)
Q(Z30424)
 
1086
CCC
GCC
197
P(L29566)
A(Z30424)
 
1273
G
A
Intron
 
 
1461
TCG
CCG
305
S(L29566)
P(Z30424)
 
1751
 GGT
GGC
401
No Change
 
1813
AAC
 GAC
419
N(L29566)
D(Z30424)
 
1889
G
C
Intron
 
 
1926
GGG
GGC
442
No change
 
1943-1944
TCT
TGC
448
S(L29566)
C(Z30424)
 
1948
TAC
AAC
450
Y(L29566)
N(Z30424)
 
2044
GCC
ACC
482
A(L29566)
T(Z30424)
 
2187
ATT
ATG
529
I(L29566)
M(Z30424)
 
2325+
-
C
Intron
 
 
2332
C
T
Intron
 
 
2343
G
T
Intron
 
 
2348
C
T
Intron
 
 
2371
TTC
TTG
Intron
F(L29566)
L(Z30424)
 
2384
GCC
CCC
578
A(L29566)
P(Z30424)
 
2416
CTG
CTC
588
No change
 

The mep gene encodes a metalloprotease ( P46075) and allergene (Aspf5). The ORF is predicted to code for 634 amino acids (Predicted MW 68 656 Da) and the mep gene contains 4 introns of 50, 51, 52 and 53 bp. A putative TATA motif is present 86 bp upstream of the start codon and a putative CAAT motif can be found 146 bp upstream of the initial methionine. The N-terminal sequence of the mature MEP protein indicates that MEP has a 245 amino acid propeptide and the extreme N-terminus of MEP has characteristics of a signal peptide. MEP contains an amino acid sequence (HEYTH) found in bacterial and eukaryotic zinc metalloproteases.

A double mep alp disruption mutant produced similar mortality levels in mice when compared to the wild type strain. This suggests that alp and mep are not essential for the invasion of lung tissue by A. fumigatus.

Aspf5 produces a mature protein of 388 amino acids and was recognized by 26 of 35 sera from allergic asthmatics without ABPA and by 50 of 54 sera from patients with ABPA. Aspf5 is able to elicit specific skin type 1 reaction in Afu-sensitized individuals. The protein shows binding capabilities to IgE after Western analysis.

Papers:

Jaton-Ogay K, Paris S, Huerre M, Quadroni R, Falchetto R, Togni G, Latge J.P, Monod M.

"Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus."

Mol. Microbiol. 14:917-928. 1994.

Aspergillus fumigates secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A, fumigates libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the 14-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous. to the active site of other bacterial and eukaryotic zinc meta lloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.

Sirakova T.D, Markaryan A, Kolattukudy P.E

"Molecular cloning and sequencing of the cDNA and gene for a novel elastinolytic metalloproteinase from Aspergillus fumigatus and its expression in Escherichia coli."

Infect. Immun. 62(10): 4208-4218. 1994.

An extracellular elastinolytic metalloproteinase, purified from Aspergillus fumigatus isolated from an aspergillosis and patient/and an internal peptide derived from it were subjected to N-terminal sequencing. Oligonucleotide primers based on these sequences were used to PCR amplify a segment of the metalloproteinase cDNA, which was used as a probe to isolate the cDNA and gene for this enzyme. The gene sequence matched exactly with the cDNA sequence except for the four introns that interrupted the open reading frame. According to the deduced amino acid sequence, the metalloproteinase has a signal sequence and 227 additional amino acids preceding the sequence for the mature protein of 389 amino acids with a calculated molecular mass of 42 kDa, which is close to the size of the purified mature fungal proteinase. This sequence contains segments that matched both the N terminus of the mature protein and the internal peptide. A. fumigatus metalloproteinase contains some of the conserved zinc-binding and active-site motifs characteristic of metalloproteinases but shows no overall homology with known metalloproteinases. The cDNA of the mature protein when introduced into Escherichia coli directed the expression of a protein with a size, N-terminal sequence, and immunological cross-reactivity identical to those of the native fungal enzyme. Although the enzyme in the inclusion bodies could not be renatured, expression at 30 degrees C yielded soluble enzyme that showed chromatographic behavior identical to that of the native fungal enzyme and catalyzed hydrolysis of elastin. The metalloproteinase gene described here was not found in Aspergillus flavus.

Aspartic protease - pep

Two research groups have cloned the pep gene:
(L31490), 1883 bp from strain isolate 13 (Lee & Kolattukudy, 1995).
(X85092), 1540 bp from strain CHUV 192 88 (Reichard et al., 1995).

Table 2. below shows the gives details of the sequence differences between L31490 and X85092 (from the start codon to the stop codon only)

Table 2.

Position (bp of L31490)
L31490
X85092
Amino acid position
Amino acid change
345+
A-T
TCT
(Frame shift) 19 - 26
MLSLWSSRA (L31490)
SAVPVVQPR (X85092)
372
C GCC AAG
CGC- AAG
 (Frame shift) 27
AK (L31490)
 RK (X85092)
388 - 389
TTC CTT
AAC CAA
33 - 34
FL (L31490)
 NQ (X85092)
803
GTC
GGC
154
G (L31490)
 V (X85092)
972+
-
C
Intron
 
1005+
-----
CGC CCT
(Frame shift) 204 - 210
 - - SDYFLY(L31490)
  RPQTTFFD(X85092)
1026+
T - AC
T GAC
(Frame shift) 211
Y(L31490)
 D(X85092)
122
ACC
TCC
274
T(L31490)
 S(X85092)
1264+
-
C
Intron
 
1483+
CC-G  TCA
CCC GTC A
(Frame shift) 345-347
SLT(L31490)
 VTD(X85092)
1494+
G-C
GGC
(Frame shift) 348-356
QLYLLRR - H(L31490)
 GSSTCYGGI(X85092)
1518+
CAT  C-AG
 C ATC CAG
(Frame shift) 357
Q (L31490)
 Q (X85092)
1533
TTG
CTG
362
No change
 
1618+
C-T
CCT
(Frame shift) 393 -396
LRHR (L31490)
  PQA-(X85092)

The pep gene codes for an aspartic protease ( P41748 Q12547) and allergene 10. The ORF is predicted to encode 395 amino acids (Predicted MW 41587.54 Da) and contains three introns of 51, 59 and 50 bp. The A. fumigatus pep gene has 60-72 % identity with aspergillopepsin genes from A. niger, A. oryzae and A. satoi. N-terminal sequencing suggests that PEP is synthesised as a zymogen with a pre-pro region of 70 amino acid residues. A putative signal peptide is found at amino acids 1 to 20. Southern analysis suggests that the pep gene is present at a single locus in the A. fumigatus genome.

Aspf10 contains 325 amino acids and a predicted molecular weight of 34.4kDa, allergene 10 is recognised by only 16 of 89 allergic aspergillosis sera tested and hence aspf10 is considered as a minor allergen of Afu . This protease has been demonstrated in the fungal elements and tissue infiltrates of fatal cases of invasive aspergillosis suggesting the production and secretion of protease in the lung tissue of infected patients.

Papers:

Lee J.D, Kolattukudy P.E.
"Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung."
Infect. Immun. 63(10): 3796-3803. 1995

Hydrolysis of structural proteins in the lung by extracellular proteinases secreted by Aspergillus fumigates is thought to play a significant role in invasive aspergillosis. This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase. We report that A. fumigates also secretes an aspartic proteinase (aspergillopepsin F) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin. The pH optimum for the enzymatic activity was 5.0 with elastinCongo red as the substrate, and the activity was not significantly inhibited by pepstatin A, diazoacetyl norleucine methylester, and 1,2-epoxy-3-(p-nitrophenoxy) propane. The cDNA and gene encoding this aspartic proteinase were cloned and sequenced. The open reading frame, interrupted by three introns, would encode a protein of 393 amino acids composed of a putative 21-amino-acid signal peptide and a 49-amino-acid propeptide preceding the 323-amino-acid mature protein. The amino acid sequence of A. fumigates aspartic proteinase has 70, 66, and 67% homology to the sequences of those from Aspergillus oryzae, Aspergillus awamori, and Aspergillus saitoi, respectively. The active-site motif (DTG) and the catalytic aspartic residues characteristic of aspartic proteinases are found in the presently described enzyme, indicating that it belongs to a family of aspartic proteinases. Polyclonal antibodies were produced in rabbits against both the mature and precursor forms of the aspartic proteinase expressed in Escherichia coli. Immunoblotting with both antibodies detected a 39-kDa mature enzyme in the culture supernatant of A. fumigates. The aspartic proteinase activity was inhibited by the antibodies, suggesting that the aspartic proteinase in the culture supernatant corresponds to the product of the cloned gene. Immunogold electron microscopy showed that the aspartic proteinase was secreted by A. fumigates invading neutropenic mouse lung and its secretion was directed toward the germ tubes of penetrating hyphae.

Reichard U, Monod M, Ruechel R.
"Molecular cloning and sequencing of the gene encoding an extracellular aspartic proteinase from aspergillus fumigatus."
FEMS Microbiol. Lett. 130:69-74. 1995

Olignucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.

 

Alkaline protease alp

Three research groups have cloned the alp gene:
( X66935) 758 bp from strain H234 ( Tang et al., 1992)
( Z11580) 2163 bp from strain CHUV 192 88 ( Jaton-Ogay et al., 1992) and
( M99420), 2930 bp ( Kolattukudy et al., 1993).

This gene has also been partially sequenced in a number of strains in an analysis of genetic variants among environmental and clinical isolates of A. fumigatus ( Katz et al., 1998).

Table 3. below gives details of the sequence differences between X66935, Z11580 and M99420 (from the start codon to the stop codon only)

Table 3.

Position (bp of Z11580)
Z11580
M99420
X66935
Amino acid position
Amino acid change
813-814
GCA
GCA
CGA
154
A(Z11580)
A(M99420)
R(X66935)
893-894 and 896
TAC AAC
TAC AAC
TAA CAA
 602-603
YN(Z11580)
YN(M99420)
TerQ(X66935)
1133+
--
CC
CC
Intron
 
1151+
-
G
G
Intron
 
1159+
-
-
G
Intron
 
1310
AC
AC
CG
Intron
 
1371-2
GCT
CGT
GCT
295
A(Z11580)
R(M99420)
A(X66935)
1477
GAT
GAT
GTT
330
D(Z11580)
D(M99420)
V(X66935)

The alp gene encodes an alkaline protease ( P28296). The ORF codes for 403 amino acids (Predicted MW 42219.44 Da) and the gene contains three introns of 57, 63 and 72 bp. N-terminal sequencing indicates that the ALP protein is processed to a mature protein of 282 amino acids (28.5 kDa). The extreme N-terminus of the ALP protein has a hydrophilic core of 9 residues suggesting the presence of a signal peptide.

An alp disruption mutant and an alp restrictocin double disruption mutant exhibited the same virulence as an A. fumigatus wild type strain in a mouse model. A double mep alp disruption mutant also produced similar mortality levels in mice when compared to the wild type strain. This suggests that alp is not involved in the virulence of A. fumigatus.

Papers:

Jayton-Ogay K, Suter M, Crameri R, Falchetto R, Faith A, Monod M.
"Nucleotide sequence of a genomic and cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus."
FEMS Microbiol. Lett. 92: 163-168. 1992. (Z11580)

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-porenzyme has a leader sequence of 121 amino acids.

Tang C.M, Cohen J, Holden D.W.
"An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity."
Mol. Microbiol. 6:1663-1671. 1992. (X66935)

Invasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus, is a life-threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAIp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 by segment of the AFAIp gene, obtained by polymerase-chain-reaction-based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAIp gene. SDS-polyacrylamide gel electrophoresis of proteins from the culture filtrate of a strain carrying the AFAIp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild-type cells, the mutant had undetectable activity on azocollagen and elastin-Congo red, over a broad pH range. This shows that AFAIp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus, and that the mutant strain will be useful in assessing the role of AFAIp in pathogenicity.

Kolattukudy P.E, Lee J.D, Rogers L.M, Zimmerman P, Ceselski S, Fox B, Stein B, Copelan E.A.
"Evidence for possible involvement of an elastolytic serine protease in aspergillosis."
Infect. Immun. 61(6):2357-2368. 1993. (M99420)

A number of isolates of Aspergillus fumigatus obtained from the hospital environment produced extracellular elastolytic activity. This activity was found to be catalysed by a single 33 kDa protein which was purified and characterized to be a serine protease. A. fumigatus, when grown on the insoluble structural material obtained from murine and bovine lung, produced the same extracellular 33 kDa elastolytic protease, indicating that this enzyme is likely to be produced when the organism infects the lung. Polymerase chain reaction with an oligonucleotide primer based on the N-terminal amino acid sequence of the elastolytic enzyme yielded a cDNA which was cloned and sequenced. The active serine motif showed more similarity to subtilisin than to mammalian elastase. The amino acid sequence showed 80% identity to the alkaline protease from Aspergillus oryzae. Screening of hospital isolates of Aspergillus flavus showed great variation in the production of elastolytic activity and a much lower level of activity than that produced by A. fumigatus. The elastolytic protease from A. flavus was shown to be a serine protease susceptible to modification and inactivation by active serine and histidine-directed reagents. This protease cross-reacted with the antibodies prepared against the elastolytic protease from A. fumigatus. Immunogold localisation of the elastolytic enzyme showed that A. fumigatus germinating and penetrating into the lungs of neutropenic mice secreted the elastolytic protease. An elastase-deficient mutant generated from a highly virulent isolate of A. fumigatus caused drastically reduced mortality when nasally introduced into the lung of neutropenic mice. All of the evidence suggests that extracellular elastolytic protease is a significant virulence factor in invasive aspergillosis.

 

 

Metalloprotease mepB

One research group has cloned the mepB gene:
( U85769) 2563 bp from strain CBS 144 89 ( Ibrahim-Granet and D'enfert et al., 1997)

The mepB gene encodes a metalloprotease ( P97996). The ORF codes for 716 amino acids (Predicted MW 81872.47 Da) and the gene contains 2 introns of 47 and 55 bp. The deduced amino acid sequence has most identity with the thimet oligopeptide family of zinc-dependent metalloproteases. MEPB contains two motifs thought to participate in catalytic activity of thimet oligopeptides, these are F(479)HELGH and D(506)FVEAP. Purified MEPB hydrolyses the P2 peptide 4-phenylazobenzlcarbonyl-pro-leu-gly-pro-arg. This is a typical substrate for the thimet oligopeptide family of metalloproteases.

Disruption of the mepB gene revealed no phenotype.

Paper:

Ibrahim-Granet O, D'Enfert C.

"The Aspergillus fumigatus mepB gene encodes a 82kDa intracellular metalloproteinase structurally related to mammalian thimet oligopeptidases."
Microbiol. 143(7): 2247-2253. 1997 (U85769).

Aspergillus fumigatus produces an 82 kDa intracellular metalloproteinase that hydrolyses the Pz-peptide, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg, a typical substrate of members of the thimet oligopeptidase family which is ubiquitously distributed across animal species. The A. fumigatus mepB gene encoding this 82 kDa metalloproteinase was cloned and sequenced. Analysis of the deduced amino acid sequence of mepB showed that the MepB protein is a cytosolic zinc metalloproteinase of the thimet oligopeptidase family (M3) and as such is probably involved in the intracellular degradation of small peptides. An A. fumigatus mutant that lacks the MepB Pz-peptidolytic activity was constructed by gene disruption at the mepB locus. Analysis of this mutant did not reveal any detectable phenotype.

Vacuolar serine protease alp2

One research group has sequenced alp2 mRNA:
( Y13338) 1787 bp from strain D141. Reichard, Monod and Ruechel, unpublished

The ALP2 protein ( P87184) is predicted to be 495 amino acids long and has a predicted MW of 52607 Da. ALP2 contains a putative signal peptide cleavage site between amino acid positions 16 and 17 and the EMBL entry suggests ALP2 is processed to a mature protein of 359 amino acid residues. Fasta analysis indicates the ALP2 protein has most identity (> 80 %) with A. niger subtilase-like serine protease and Penicillium citrinum alkaline serine protease. A profile scan using the ALP2 sequence revealed sequences from the subtilase family of serine proteases. Subtilases usually have three active site signatures based around Asp, Ser and His residues and the profile scan for ALP2 revealed an Aspartic acid active site (residues 154-212), a Serine active site (residues 193-242) and a Histidine active site (residues 356-409). The profile scan also revealed an ATP/GTP-binding site A motif at residues 194-201.

Paper:

Reichard U, Cole G.T, Hill T.W, Reuchel R, Monod M.
"Molecular characterisation and influence on fungal development of Alp2, a novel serine protease from Aspergillus fumigatus."
Int. J. Med. Microbiol. 290: 549-558. 2000 (AFU243145 / Y13338)

A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, Int. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries. ALP2 shares a 49 % identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae. In addition there is a 78 % identity with PEPC, a serine proteinase which has been described in Aspergillus niger. Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80 % reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50 % reduction of the median diameter of conidiophore vesicles. The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A. fumigatus.

Aspartic protease pep2

One research has cloned the pep2 gene:
( AFU132504) 2300 bp from strain D141. Reichard U., Monod M., Reinhard R., unpublished

The pep2 gene has three putative introns of 88, 60 and 60 bp. The PEP2 protein ( O42630) is predicted to be 398 amino acids long and has a predicted molecular weight of 43354 Da. PEP2 contains a putative signal peptide cleavage site between amino acid positions 17 and 18. The EMBL entry indicates the PEP2 protein is processed to a mature peptide of 327 amino acids. Fasta analysis indicates that PEP2 has most identity (87 %) with the A. niger aspartic protease PEPE. A profile protein database scan revealed a eukaryotic and viral aspartyl proteases active site signature sequence between residues 76 and 397.

Paper:

Reichard U, Cole G.T, Ruechel R, Monod M.
"Molecular cloning and targetted deletion of pep2 which encodes a novel aspartic proteinase from Aspergillus fumigatus."
Int. J. Med. Microbiol. 290: 85-96. 2000 (AFU132504 / AFY15744)

An aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes. PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis.