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Lysosomal Protective Protein

(Cathepsin A)
Overview
Lysosomal protective protein (Cathepsin A) is a protein which associates with galactosidase and neuraminidase to protect them from degrading, thus promoting their ability
to catabolise oligosaccharides. Cathepsin A also has a role as a protease, breaking down
other proteins. It most often acts in cellular lysosomes (Hiraiwa, 1999 and Itoh et. al, 1997).
Lysosomal protective protein is encoded by the CTSA gene on human chromosome twenty.
Defects in this gene lead to the disease galactosialidosis (Okamura-Oho, Zhang & Callahan,
1994).

Disease
Mutations in the CTSA gene, encoding Lysosomal Protective Protein (Cathepsin A), result in
galactosialidosis. The disease is broadly classified into early infantile, late infantile and
juvenile/adult forms. Early infantile is characterised by onset at or around birth, oedema,
ascites (excess fluid between the tissues lining the abdomen and organs), bone deformities,
coarse distinctive facial expressions and hepatosplenomegaly (enlargement of the spleen and
liver). Late infantile can be separated into cardiac-dominant if cardiac complications are
prominent or neurological-dominant if neurological symptoms predominate. Skeletal
dysplasia, conductive hearing loss, hepatosplenomegaly and inguinal hernias were also
common in cardiac-dominant while mental retardation, seizures, acoustic myoclonus and
cherry-red spots are often observed in neurological-dominant. For juvenile/adult, the main
symptoms include neurological abnormalities, skeletal dysplasia and cardiac complications
(Okamura-Oho, Zhang & Callahan, 1994). Oligosachcharide levels in urine can be used for
pre-diagnosis of galactosialidosis, though specific blood or skin biopsy tests for decreased -

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galactosidase and neuraminidase levels can be used. These levels can also be detected via
amniocentesis or chorionic villus sampling. If a family history of a certain gene mutation is
present, Lysosome protective protein gene detection can be used (Itoh et. al, 1997 and
Wenger, Coppola & Liu, 2003). There is currently no cure for galactosialidosis and instead
treatment focuses on treating secondary issues and conducting routine follow-ups (Murthy et.
al, 2010). Lysosomal protective protein is responsible for stabilising -galactosidase and
neuraminidase complexes within cells. It also has a proteolytic function. Cathepsin A in
conjunction with -galactosidase and neuraminidase is responsible for breaking down
oligosaccharides. Cathepsin A also protects -galactosidase and neuraminidase from being
catabolised in a cells lysosome. Dysfunction of Cathepsin A leads to an increase in the
levels of oligosaccharides and a decrease in -galactosidase and neuraminidase levels, further
increasing oligosaccharide levels. The oligosaccharides progressively build-up and gradually
cause the symptomatic presentation of galactosialidosis (Hiraiwa, 1999 and Itoh et. al, 1997).

Affected gene
Cathepsin A (CTSA) is the gene responsible for producing Lysosomal protective protein and
which, when defective, results in the symptoms of galactosialidosis. The CTSA gene has 15
exons and 14 introns with various regulatory elements upstream and within the gene (Figure
1). It is located on Chromosome 20, q13.1 (National Centre for Biotechnology Information,
<http://www.ncbi.nlm.nih.gov/nuccore/NG_008291.1>). There are 70 known orthologues of the
human CTSA gene, including the Orangutan, Chimpanzee, Gorilla and Macaque, which
share the most likeness to the human gene. All except the Macaques are located on the
animals chromosome 20. There are two paralogues, CPVL (vitellogenio-like
carboxypeptidase) on chromosome seven and SCPEP1 (serine carboxypeptidase 1) on
chromosome seventeen, which are both apart of the carboxypeptidase family, like CTSA
(Ensembl,<http://asia.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000064601;r

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=20:44519591-44527459;t=ENST00000372484>). Fifteen allelic variants of the CTSA gene are

known to cause galactosialidosis. The type of mutation can reflect the severity of
galactosialidosis and the time of onset. Allelic variant 0.0001 causes late infantile
galactosialidosis and involves a T-to-G transversion in the gene at position 1324, which
results in the change from valine to phenylalanine in the resulting protein. A common
mutation that is involved in many allelic variants causing galactosialidosis involves an A-toG transition in the intron after exon seven, resulting in the skipping of exon seven when
transcribed. This mutation may be present in conjunction with other mutations as well, as
with allelic variant 0.003, which involves the intron seven splice site mutation and an A-to-G
transition in exon two, transforming a glutamine to arginine in the protein. There are many
splice variants, which produce different protein isoforms
(Ensembl,<http://asia.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000064601;r
=20:44519591-44527459;t=ENST00000372484> and Online Mendelian Inheritance in Man,

<http://omim.org/entry/613111>).

Figure 1: CTSA gene and associated mRNA and exon structure

Affected protein
The protein affected in galactosialidosis is Lysosomal protective protein (Cathepsin A). It
functions to cleave active protein hormones including Endothelin I, Angiotensin I,
Bradykinin, Oxytosin and Vasopressin (ADH), using a serine protease catalytic centre. This
ultimately changes the bioactivity of the active protein. Cathepsin A does this by
carboxypeptidase, deaminase and esterase actions. One such example is its function as a

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deaminase by catalysing the hydrolysis of amine bonds for tachykinin peptides. It also
performs an intra-lysosomal protective role for -galactosidase and neuraminidase, ensuring
that they are not degraded by lysosomes (hence, its name) when it incorporates into a multiprotein complex with them. Cathepsin A is present in high concentrations in kidney, liver
and lung and lower concentrations in the brain. (Hiraiwa, 1999). The 108kDa dimer form
(two 54kDa monomers each containing two polypeptides, a 32kDa and a 20kDa) of
Cathepsin A, its mature state for lysosomal activity, consists of two cap domains and two
core domains, one for each monomeric unit. It also contains a 2kDa maturation site that is
excised to produce the active protein, as shown in Figure 2. The entire protein is 480 amino
acids long.
Figure 2: Cartoon structure of Cathepsin A, illustrating core and cap domains. The excised peptide is shown in blue.

It is a member of the serine protease family

of proteins, containing a serine triad for its
catalytic site. It is classified as a
carboxypeptidase because it cleaves other
proteins from the carboxy end of the protein
(exopeptidase activity). Historically, it is
grouped with other lysosomal proteins in the
Cathepsin group because it is a protease
with similar human history to other
cathepsins. It is also proposed to be included in the / hydrolase family, a group of
structurally similar proteins that all contain eight sheets joined by helicies. They also
contain a catalytic triad within the conserved structure. This group of proteins do not
necessarily share sequence homology, but share an overall structural similarity. (Hiraiwa,
1999). Sequence similarity is shown as a phylogenic tree in Figure 3. The tree compares
Cathepsin A with Carboxypeptidase Y, an orthologue in yeast that shows close sequence

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homology, human Lipase, a member of the / hydrolase family, human Cathepsin G,

another cathepsin protein, human Factor Xa/prothrombinase, a member of the serine protease
family like Cathepsin A, and Hexokinase 2, a common human enzyme used as an out group
for comparison. Cathepsin A shares the most homology with yeast Carboxypeptidase Y
(0.72), followed by Cathepsin G (0.94), then human Hexokinase 2 (0.96), the serine protease

Figure 3: A phylogenetic tree of Cathepsin A showing relationships with a yeast orthologue, Carboxypeptidase Y, another cathepsin (Cathepsin
G), a member of the / hydrolase family (Lipase), another serine protease (FactorXa) and a common human enzyme (Hexokinase 2) for
comparison.

human Factor Xa (0.97), and least homology with the / hydrolase family human Lipase
(0.98) (ClustalW2, http://www.ebi.ac.uk/Tools/msa/clustalw2/>). The complex threedimensional structure of Cathepsin A consists of multiple alpha helicies and beta sheets
joined by loops, as shown in Figure 4. It contains two chains arranged by PyMOL into eight
distinct regions, as illustrated by different colours in Figure 4. The excision peptide (shown
in magenta in Figures 4, 5 and 6) plays an integral part in the maturation of the Cathepsin A
protein, allowing the catalytic triad to become active in order to cleave its target proteins.
Cathepsin As catalytic triad is highlighted in red in Figure 5. It consists of residues 150Ser,
372

Aspartate and 429Histidine. The complex three-dimensional structure of cathepsin A is

stabilised by hydrophobic interactions and disulphide bonds. The four disulphide bonds
linking chains of cathepsin A are shown in yellow in Figure 6. The 60Cys334Cys disulphide
bond is especially important for ensuring correct structural formation when binding
substrates.
There are more than ten mutations in the Cathepsin A gene (CTSA) that are known to cause
galactosialidosis. Often, patients are heterozygous for two or more mutations within the gene

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(Hiraiwa, 1999). The juvenile form of galactosialidosis is most commonly associated with a
mutation in the CTSA gene intron seven splice donor site, causing the skipping of exon seven
during transcription. This removes a section of the Cathepsin A protein, disallowing it to
function correctly. The late infantile form most commonly has mutations in 412Phe and
221

Tyr, affecting key secondary structural elements (a -sheet for 412Phe and a joining

segment for 221Tyr) within the protein. The Phenylalanine mutant in particular may hinder
the formation of the Cathepsin A dimer, which is needed to complex with neuraminidase and
-galactosidase. The early infantile form is associated with substitutions that prevent the
formation of the Cathepsin A precursor, reflecting the clinical severity of this form. One
such example is a Japanese family with a Tyr367Cys substitution (Hiraiwa, 1999, Shimmoto
et. al, 1993, and Zhou et. al, 1993).

Figure 4: PyMOL structure of Cathepsin A (1IVY) showing the different chains that comprise the secondary
structures of the protein, with the excision peptide shown in magenta and catalytic triad selected with purple dots.

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Figure 5: A close-up of the three residues that make up Cathepsin As catalytic triad, shown in red. Excision peptide
is shown in magenta.

Figure 6: Cathepsin A with disulphide bonds shown in yellow.

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Summary:
Lysosomal protective protein/Cathepsin A is a protein present intra- and extra-lysosomally
that functions to protect the activity of neuraminidase and -galactosidase when complexed
with these proteins as well as cleave small bioactive peptides using its serine protease
activity. Defects in the gene encoding this protein (CTSA) are known to cause
galactosialidosis, a disease characterised by bone abnormalities, cardiac complications,
enlarged spleen and liver and neurological deficits. It is divided into three clinical
presentations: juvenile, late infantile and early infantile, each of which have a subset of
mutations known to cause the subtype of galactosialidosis and which have specific symptoms
associated with them. The protein is composed of 480 amino acids that folds into a complex
3d structure consisting of many alpha helicies and beta sheets. The salient features of its
structure include: arrangement into two separate chains, a serine protease catalytic triad, four
disulphide bonds and an excision peptide in the zymogen form. More research is needed to
understand the complex interactions between ligands such as neuraminidase and galactosidase and the role that mutations in the CTSA gene contribute to specific subsets of
galactosialidosis. In particular, structural and functional information about the complex of
cathepsin A, neuraminidase and -galactosidase would shed light onto how cathepsin A plays
a role in protecting these important lysosomal proteins. Further research would also enable
novel therapies to be developed for therapeutic use.

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References
ClustalW2 2011, European Bioinformatics Institute, 18 September, 2011,
<http://www.ebi.ac.uk/Tools/msa/clustalw2/>.
Ensembl 2011, European Bioinformatics Institute and Wellcome Trust Sanger Institute,
viewed 15 August, 2011,
<http://asia.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000064601;r=20:
44519591-44527459;t=ENST00000372484>.

Hiraiwa, M 1999, Cathepsin A/protective protein: an unusual lysosomal multifuncational

protein, Cell and Molecular Life Sciences, vol. 56, pp. 894-907
Itoh, K, Miharu, N, Ohama, K, Mizoguchi, N, Sakura, N & Sakuraba, H 1997, Fetal
diagnosis of galactosialidosis (protective protein/Cathepsin A), Clinica Chimica Acta,
vol. 266, pp. 75-82
Murthy, TE, Nagarjuna, S, Vali, P, Saritha, T & Rao, G 2010, International Journal of
PharTech Research, vol. 2, no. 2, pp. 1082-1091
National Centre for Biotechnology Information 2011, United States National Library of Medicine,
viewed 15 August, 2011, <http://www.ncbi.nlm.nih.gov/nuccore/NG_008291.1>.

Okamura, Y, Zhang, S, Callahan, JW 1994, The biochemistry and clinical features of

galactosialidosis, Biochimica et Biophisica Acta, vol. 1225, pp. 224-254
Online Mendelian Inheritance in Man 2009, John Hopkins University, viewed 15 August,
2011, <http://omim.org/entry/613111>.
Shimmoto, M, Fukuhara, Y, Itoh, K, Oshima, A, Sakuraba, H & Suzuki, Y 1993, Protective
protein gene mutations in galactosialidosis, Journal of Clinical Investigation, vol. 91,
pp. 2392-2398.
Wenger, DA, Coppola, S & Liu, S 2003, Insights into the diagnosis and treatment of
lysosomal storage diseases, Archives of Neurology, vol. 60, pp. 322-328

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Zhou, XY, van der Spoel, A, Rottier, R, Hale, G, Willemsen, R, Berry, GT, Strisciuglio, P,
Morrone, A, Zammarchi, E, Andria, G & dAzzo, A 1996, Molecular and biochemical
analytics of protective protein/cathepsin A mutations: correlation with clinical severity
in galactosialidosis, Human Molecular Genetics, vol 5, pg 1977-1987.