Transcript Alkaptonuria and Aspergillus nidulans

The use of Aspergillus nidulans to
identify the genetic defect responsible
for the human metabolic disorders
Alkaptonuria and Type I Tyrosinemia.
A slide set provided by Prof Miguel A Penalva
Centro de Investigaciones Biologicas del CSIC, Madrid 28006, Spain.
Aspergillus nidulans –a useful genetic model.
Alkaptonuria is a rare genetic disorder.
The discovery of the gene responsible for alkaptonuria and a
number of genes responsible for human inborn errors of
metabolism, have exploited the mold Aspergillus nidulans.
This fungus has the experimental advantages of:
• rapid growth
• easy genetic manipulation
• growth requirements that are readily manipulated
• this filamentous fungi happens to share the same pathway of
aromatic amino acid metabolism as humans, lending itself
exquisitely to their study.
Symptoms of alkaptonuria
Normal urine
Urine from patients
with alkaptonuria
Patients may display painless bluish darkening of the outer ears,
nose and whites of the eyes. Longer term arthritis often occurs.
Homogentisic acid is an intermediate in the degradation pathway of
phenylalanine. The reaction is catalysed by homogentisate dioxygenase
(HGO).
homogentisic acid
OH
HOOC
CH
OH
HGO
CH2
CH
O
O
C
C
CH2 CH2
COOH
maleylacetoacetic acid
COOH
A deficiency of HGO causes
alkaptonuria.
Catabolic pathway for phenylalanine and
tyrosine
Defect here causes
alkaptonuria
Defect here causes
Type I Tyrosinemia
Homogentisate
dioxygenase
Fumarylacetoacetate
hydrolase
Characterisation of genes of the phenylalanine/ PhAc
degradation pathway
Genes encoding catabolic pathway
enzymes are strongly induced by the
pathway substrate(s), facilitating their
cloning by subtractive cDNA
hybridization.
No transcripts were detected when
tissue is grown in glucose only.
Common cDNA's are subtracted out
from the target population by
hybridisation to RNA from tissue grown
in the absence of phenylalanine, the
target cDNA is thus enriched for the
transcript which has been induced by
the presence of its substrate
phenylalanine.
Characterisation of genes of the phenylalanine/PhAc
degradation pathway
Using a subtractional cDNA cloning
strategy for A.nidulans to obtain
phenylalanine degradation genes and
subsequently their derived protein
sequences, it was possible to screen
the human databases in search of
cDNAs of human homologues.
The first fungal gene obtained was fahA encoding a
protein FAAH (fumarylacetoacetate hydrolase).
Screening showed a 47% homology with human
FAAH. This enzyme catalyses the last step in
catabolism of phe and tyr.
47 % identity
This was significant evidence to imply that fungi
and humans share the same catabolic pathway for
the breakdown of phe and tyr aromatic rings.
homogentisic acid
C
C
CH2 CH2
CH
COOH
O
O
=
CH
O
=
HOOC
O
FAAH catalyses the last step in the
degradation path of phenylalanine and
tyrosine.
HOOC - CH2 - CH2 - C - CH2 - C - CH2 - COOH
maleylacetoacetic acid
succinylacetoacetic acid
spontaneous
HOOC
CH
CH2 CH2
CH
O
O
fumarylacetoacetate
FAAH
O
O
=
C
=
C
COOH
COOH
HOOC - CH2 - CH2 - C - CH2 - C - CH3
Succinylacetone
toxic and mutagenic
Fumarate + acetoacetate
Deficiency of the enzyme FAAH results in Type I tyrosinemia
Tyrosinemia is diagnosed by a blood and urine test.
Tyrosinemia is treated by a low protein diet (low in
phenylalanine, methionine and tyrosine) and a drug
called NTCB.
WHAT ARE THE SYMPTOMS OF TYROSINEMIA?
The clinical features of the disease ten to fall into two
categories, acute and chronic.
In the so-called acute form of the disease, abnormalities
appear in the first month of life. Babies may show poor weight
gain, an enlarged liver and spleen, a distended abdomen,
swelling of the legs, and an increased tendency to bleeding,
particularly nose bleeds. Jaundice may or may not be
prominent. Despite vigorous therapy, death from hepatic
failure frequently occurs between three and nine months of
age unless a liver transplantation is performed.
But the search for the defective enzyme
responsible for alkaptonuria was still on.
•In TBLASTIN searches of the human EST
database the amino acid sequence of
fungal HGO identified candidate cDNAs for
human HGO.
•Most of these ESTs, which represent a
single transcript, came from liver cDNA
libraries.
•The human protein encoded by this
transcript showed 52% homology to
fungal HGO and when expressed in
bacteria it gave HGO activity.
A fungal mutant strain (DfahA) deficient in the enzyme
FAAH was unable to grow on phenylalanine alone. When
lactose was also present the fungus strain still did not grow
DfahA
fahA+
Lactose
phenylalanine
No colony
growth in
mutant
strain of
A.nidulans
Lactose +phenylalanine
Then further colonies of the FAAH deficient strain
developed which had resistance to the toxic
phenylalanine.
It was thought that other enzyme mutations
upstream from FAAH were responsible.
Wild type
Phe
Tyr
DfahA
DfahA
hmgA-
pHPP
HGA
lactose
Lac + Phe
Phe
MAA
FAA
The double mutant colonies
were
able to grow in the presence of
F + AcAc
phenylalanine and secreted a
brown pigment identified as
homogentisate.
Accumulation of ochronotic pigment in the culture supernatant
of the ‘alkaptonuric’ fungus
Alkaptonuria enjoys the
historic distinction of being
one of the conditions for
which recessive inheritance
was first proposed.
This proposal was made in 1902 by the English
physician Archibald Garrod. In a series of brilliant
lectures in 1908 Garrod set forth the charter group of
what he called "inborn errors of metabolism."
The 4 conditions he labelled as inborn errors were
albinism, cystinuria, pentosuria and, of course,
alkaptonuria.
These slides were adapted from material
kindly provided by Miguel A Penalva,
Centro de Investigaciones Biologicas del
CSIC, Madrid 28006, Spain.
With acknowedgement of the following people;
Santiago Rodríguez de Córdoba
Magdalena Ugarte
David Timm
José M. Fernández Cañón
José M. Rodríguez Rodríguez