The document discusses protein glycosylation and its role in muscular dystrophies. It provides background on the dystrophin-associated protein complex (DAPC) and how mutations that affect its glycosylation can cause various forms of muscular dystrophy. Specifically, it describes how mutations in glycosyltransferase genes involved in glycosylating α-dystroglycan, an important component of the DAPC, lead to hypoglycosylation of α-dystroglycan and muscular dystrophy phenotypes. One such disease is Fukuyama-type congenital muscular dystrophy, caused by mutations in the fukutin gene that encodes a glycosyltransferase thought to play a role in α-dystrogly
Protein glycosylation and its associated disordersSaranya Sankar
Protein glycosylation and its associate disorders. Glycosylation is one of the post translational modifications important for the normal function of the protein such as cell adhesion, signalling etc.. defect in this process leads to fatal disorder such as cancer, PNH....
Protein glycosylation and its associated disordersSaranya Sankar
Protein glycosylation and its associate disorders. Glycosylation is one of the post translational modifications important for the normal function of the protein such as cell adhesion, signalling etc.. defect in this process leads to fatal disorder such as cancer, PNH....
What is Glycoprotein ?:
Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains.
This process is known as glycosylation.
The carbohydrate is attached to the protein during the following modifications: Co-translational modification & Post-translational modification.
In proteins that have segments extending extracellularly, the extracellular segments are often glycosylated.
Proteoglycans are protein chains that are covalently bonded at multiple sites to a class of polysaccharides, known as glycosaminoglycans.Glycosaminoglycans constitute 95% of proteins.Proteoglycans are synthesised in RE and transported to GA where they are modified in to various forms.Proteoglycans are major component of ECM and their role is depended on the type of GAGs they associate with.
INTRODUCTION
STRUCTURE
TYPES OF BONDS
N-LINKED GLYCOSYLATION
O-LINKED GLYCOSYLATION
AMOUNT OF CARBOHYDRATES PRESENT IN GLYCOPROTEIN
BIOLOGICAL SIGNIFICANCE AND FUNCTION
I CELL DISEASE
BIOLOGICAL ADVANTAGE OF ADDING OLIGOSACCHARIDES TO PROTEIN
CONCLUSION
REFERENCES
Glycoprotein, lipoprotein, structure and functionKAUSHAL SAHU
INTRODUCTION
DEFINITION
STRUCTURE
TYPES OF BONDS
N-LINKED GLYCOSYLATION
O-LINKED GLYCOSYLATION
AMOUNT OF CARBOHYDRATES PRESENT IN GLYCOPROTEIN
BIOLOGICAL SIGNIFICANCE AND
FUNCTION
BIOLOGICAL ADVANTAGE OF ADDING OLIGOSACCHARIDES TO PROTEIN
Introduction
Protein modifications
Folding
Chaperon mediated
Enzymatic
Cleavage
Addition of functional groups
Chemical groups
Hydrophobic groups
Proteolysis
Conclusion
Reference
What is Glycoprotein ?:
Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains.
This process is known as glycosylation.
The carbohydrate is attached to the protein during the following modifications: Co-translational modification & Post-translational modification.
In proteins that have segments extending extracellularly, the extracellular segments are often glycosylated.
Proteoglycans are protein chains that are covalently bonded at multiple sites to a class of polysaccharides, known as glycosaminoglycans.Glycosaminoglycans constitute 95% of proteins.Proteoglycans are synthesised in RE and transported to GA where they are modified in to various forms.Proteoglycans are major component of ECM and their role is depended on the type of GAGs they associate with.
INTRODUCTION
STRUCTURE
TYPES OF BONDS
N-LINKED GLYCOSYLATION
O-LINKED GLYCOSYLATION
AMOUNT OF CARBOHYDRATES PRESENT IN GLYCOPROTEIN
BIOLOGICAL SIGNIFICANCE AND FUNCTION
I CELL DISEASE
BIOLOGICAL ADVANTAGE OF ADDING OLIGOSACCHARIDES TO PROTEIN
CONCLUSION
REFERENCES
Glycoprotein, lipoprotein, structure and functionKAUSHAL SAHU
INTRODUCTION
DEFINITION
STRUCTURE
TYPES OF BONDS
N-LINKED GLYCOSYLATION
O-LINKED GLYCOSYLATION
AMOUNT OF CARBOHYDRATES PRESENT IN GLYCOPROTEIN
BIOLOGICAL SIGNIFICANCE AND
FUNCTION
BIOLOGICAL ADVANTAGE OF ADDING OLIGOSACCHARIDES TO PROTEIN
Introduction
Protein modifications
Folding
Chaperon mediated
Enzymatic
Cleavage
Addition of functional groups
Chemical groups
Hydrophobic groups
Proteolysis
Conclusion
Reference
Proteoglycans are proteins that are heavily glycosylated*. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan (GAG) chain(s).
Corticosteroid induced disorders – An overviewpharmaindexing
Glucocorticoids are important in the treatment of many inflammatory, allergic, immunologic, and malignant disorders, and the toxicity of glucocorticoids is one of the commonest causes of iatrogenic illness associated with chronic inflammatory disease.Glucocorticoid-induced muscle atrophy is characterized by fast-twitch or type II muscle fiber atrophy. Corticosteroid (CS) therapy is widely used in the treatment of rheumatic diseases.Osteoporosis remains one of its major complications.Steroid induced glaucoma is a form of open angle glaucoma occurring as an adverse effect of corticosteroid therapy. Glucocorticoids induce hepatic and extrahepatic insulin resistance.Glucocorticoid treatment impairs both glucose transport in fat and muscle cells. Corticosteroid-induced psychosis represents a spectrum of psychological changes that can occur at any time during treatment. Cushing’s syndrome describes the signs and symptoms associated with prolonged exposure to inappropriately high levels of the hormone cortisol. Physicians must be aware of these adverse effects and be equipped to manage them.
Abstract
Mitogen-Activated Protein Kinase (MAPK) pathway is a signal transduction pathway that functions in a wide range of physiological and pathophysiological cellular events including cell proliferation, differentiation, apoptosis, migration, inflammation, metabolic disorders and diseases. In skeletal muscle, it plays an essential role in muscle fiber specialization, muscle mass maintenance, damage induced muscle regeneration and muscle diseases. This review provides an overview of MAPK pathway and its pathophysiological role in skeletal muscle diseases with a primary focus on muscular dystrophy and atrophy.
Similar to Protein Glycosylation and its Role in Muscular Dystrophies (20)
Protein Glycosylation and its Role in Muscular Dystrophies
1. 1
Protein Glycosylation and its Role in the Muscular
Dystrophies
Name: James Franklin, Year: 2011-2012, Degree: Biology, Supervisor: Philip
Williamson
Word Count (not including references): 5127
Abstract
The dystrophin associated protein complex plays an extremely important role in
muscle tissues. It bridges the intracellular cytoskeleton in muscle cells to
extracellular matrix components such as laminin-2. When components of this
complex are mutated and missing, the links between the cells in muscle fibres
break down, leading to the muscle wasting and weakening phenotype known as
muscular dystrophy. Alpha-dystroglycan is a very important component of the
complex, as it is responsible for binding to the extracellular matrix via its
numerous sugar chains. Mutations in glycosyltransferases that serve to
glycosylate α-dystroglycan result in hypoglycosylation of the complex, preventing
α-dystroglycan from binding to the extracellular matrix. This causes a similar
phenotype seen in the more standard muscular dystrophies. Glycosyltransferases
involved in α-dystroglycan glycosylation include fukutin, fukutin-related protein,
POMGnT1, POMT1/2 and LARGE, which can causes diseases such as fukuyama-
type muscular dystrophy, limb girdle muscular dystrophy, MDC1C, muscle-eye-
brain disease, Walker-Warburg syndrome and LARGEmyd mouse. I aimed to give
an overview of all these glycosyltransferases and their associated diseases, as
well as explore numerous potential genotype-phenotype linkages that can be
made within them. Whilst massive progress in dystroglycanopathy has been made
over the years, there is still a lot we don’t yet know.
Introduction
For a long time, the functions of sugar chains on proteins were thought to be of little
relevance. This coupled with the difficulty of actually studying the structures of these sugars
made the field of glycobiology seemrelatively unimportant. However, when sugar chains on
cell surface proteins were revealed to have a possible function in cell-cell recognition in
1970, glycobiology became a relevant subject for study (Kobata, 1992) and encouraged the
development of methods to examine these carbohydrate structures in detail.
Numerous diverse functions have been shown to exist for glycoconjugates, including
structural roles within and between cells, the ensuring of proper protein folding and
stability, the providing of target sites for microorganisms, toxins and antibodies and the
masking of these target sites, and the activation and deactivation of various protein
functions (Varki, 1993). When the DNA encoding for a protein becomes mutated such that
2. 2
the genomic code changes, the protein produced will have a different amino acid sequence
and become misfolded. A misfolded protein will be unlikely able to carry out its intended
function as the structure of its active sites will have changed such that it will no longer be
able to bind to its target sites. Mutations in proteins involved in protein glycosylation often
cause secondary defects in other proteins that depend on themselves receiving proper
glycosylation to function. For example, the protein pro-papain requires proper glycosylation
before it is able to be secreted (Vernet et al. 1990).
In this essay I will be focusing on α-dystroglycan, a heavily glycosylated protein which forms
a major part of the dystrophin associated protein complex (DAPC). Mutations directly
affecting proteins within this complex result in many different types of muscular dystrophy.
In addition, indirect mutations resulting in the hypoglycosylation of α-dystroglycan also
present its share of muscular dystrophy phenotypes (Martin-Rendon & Blake, 2003), in
addition to various brain and eye malformations. In this essay, I will discuss how mutations
in various glycosyltransferases, proteins involved in the glycosylation of proteins can lead to
the improper function of α-dystroglycan and the resulting types of muscular dystrophy.
Glycosylation Basics
There exist two main types of protein glycosylation that occur in nature. The first and most
common type is N-linked glycosylation, in which the oligosaccharide Glc3-Man9-GlcNAc2 is
added (most commonly) to an asparagine residue on a novel protein in the endoplasmic
reticulum (ER). The second, less common type is O-linked glycosylation, usually involving the
addition of an N-acetyl-galactosamine (GalNAc) sugar to either a serine or threonine residue
in the Golgi apparatus.
All types of N-linked glycans are built upon the same pentasaccharide known as the
trimannosyl core (Kobata, 1992). The core comprises a mannose bound by two other
mannose residues and to N-acetylglucosamine (GlcNAc), which is bound to another GlcNAc
residue that is attached to the protein itself (summarised in Figure 1). Upon this common
core three main types of glycan structure can exist; complex, high-mannose and hybrid.
Complex type N-linked glycans contain no other mannose sugars than those found in the
trimannosyl core. Many different components can be found attached to the outer mannose
residues of complex N-linked glycans, including GlcNAc, GalNAc, galactose, fucose, sialic
acids, and sulphates. High-mannose N-linked glycans are so called because only mannose
residues are found attached to the trimannosyl core. A common heptasaccharide is formed
from the two branching mannose residues, shown in Figure 2. Variation is created in high-
mannose glycans through the different positions and amount of additional mannose
residues attached to this common heptasaccharide structure (Kobata, 1992). Hybrid N-
linked glycans are made up of the structural features seen in both the complex and high-
mannose types. They normally have the same basic structure as seen in the common
heptasaccharide of high-mannose N-linked glycans (Figure 2), with the addition of the
3. 3
different residues found in the complex N-linked glycans bound to the unbranched outer
mannose residue.
Figure 1. The basic structure of the trimannosyl core. Key: Asparagine, blue pentagon;
GlcNAc, red square; green circle, mannose.
Figure 2. Most common structure of mannose-rich glycans. Key: Asparagine, blue pentagon;
GlcNAc, red square; green circle, mannose.
In comparison to N-linked glycans, O-linked glycans show fewer structural rules (Kobata,
1992). Because of this, it is somewhat harder to concisely define the types of O-linked
glycans, but generally they are formed in a step-by-step fashion through the addition of
monosaccharide units to serine or threonine residues. The initial monosaccharide units
bound to the serine/threonine can either be GalNAc, GlcNAc, fucose, glucose, or mannose
sugars, depending on the protein being glycosylated. Additional carbohydrates can be
bound to these initial sugars to form the rest of the oligosaccharide unit. O-linked
glycosylation is especially significant in numerous muscular dystrophies, as the protein α-
dystroglycan is heavily O-glycosylated. A-dystroglycan is a major component of the DAPC
and plays extracellular adhesion roles through its binding with numerous components of the
extracellular matrix (ECM).
The Dystrophin Associated Protein Complex
The DAPC is a protein complex made up of numerous protein components, most abundantly
found on the sarcolemma of muscle cells (Figure 3). The DAPC forms an interface that allows
the inner cell cytoskeleton and ECM to attach to each other. Mutations in many of these
components can lead to various forms of muscular dystrophy which all share common
4. 4
symptoms of muscle weakness and degeneration (Davies & Nowak, 2006). The DAPC is
thought to protect the sarcolemma from mechanical damage through the turbulant nature
of muscle fibre contraction. Therefore, as members of the DAPC become mutated and
missing, the overal sarcolemma becomes significantly weakened resulting in progressive
muscle fibre damage over time.
Figure 3. The DAPC links the ECM and the intracellular cytoskeleton via its various protein
components. Taken from Rendon & Blake 2003.
The dystrophin protein itself exists in the sarcoplasmand is what binds the actin
cytoskeleton to the rest of the DAPC. It is an elastic protein thanks to its triple helical repeat
structure, which probably acts to protect the muscle cell from mechanical damage caused
through muscle contraction (Pasternak et al. 1995). Dystrophin binds to the dystroglycans
through its cysteine-rich domain and to α-dystrobrevin via its C-terminus. Dystroglycan is
post-translationally cleaved into α and β subunits, which remain non-covalently linked
within the DAPC (Ibraghimov-Beskrovnaya et al. 1992). β-dystroglycan remains nestled in
the sarcolemma whilst α-dystroglycan binds to ECM proteins such as laminin-2, agrin and
neurexin through its O-linked glycans. Dystrophin also binds two syntrophin molecules, α1
and β1, which are thought to localize signalling molecules to create a signal-transduction
complex within the DAPC (Oak et al. 2001). The sarcoglycan complex also forms a major part
of the DAPC, and consists of a β and δ-sarcoglycan core bound by α and γ-sarcoglycan. The
sarcoglycan complex binds to α-dystroglycan via biglycan, and also binds the
transmembrane protein sarcospan, which allows the sarcoglycan members to form a single
functional unit (Hayashi et al. 2006).
5. 5
Overall, the DAPC is a complicated mesh of proteins which all appear to serve a function.
Often times when one of these proteins becomes mutated, it results in the rest of the DAPC
not forming correctly and causing some kind of muscular dystrophy. For example, Duchenne
MD, the most common form of muscular dystrophy, is caused by a mutation in the
dystrophin gene, resulting in complete loss of the dystrophin protein. Another mutation of
dystrophin, Becker MD, causes a much milder form of muscular dystrophy, as a truncated
dystrophin protein is still produced and able to serve some reduced function (Koenig et al.
1988). This theme of causing a more severe muscular dystrophy phenotype as components
become reduced also applies to glycosylation.
Diseases of α-dystroglycan Glycosylation
A group of muscle disorders known as congenital muscular dystrophies (CMDs), muscular
dystrophies present from birth, all result in the hypoglycosylation of α-dystroglycan on the
surface of muscle cells. It’s these protein linked glycans which are responsible for the
binding of the muscle cell to ECM proteins such as laminin-2, agrin and neurexin. When α-
dystroglycan is improperly glycosylated, the link between the muscle cell sarcolemma and
the muscle basal lamina is interrupted, causing the muscular dystrophy phenotype (Esapa et
al. 2002), and depending on the nature of the disease phenotype, various brain and eye
symptoms as well. The muscular dystrophy phenotype is mirrored in mutations involving
laminin-2 (Ervasti & Campbell, 1993). This is because the defective laminin-2 is unable to
bind to α-dystroglycan, resulting in the same muscular dystrophy phenotype caused by
hypoglycosylated α-dystroglycan; disruption between the muscle fibres and overall muscle
weakening.
The binding of α-dystroglycan to the extracellular matrix was proven experimentally
(Michele et al. 1995), in which α-dystroglycan purified from patients suffering from various
glycosylation disorders had significantly reduced affinity for laminin, agrin and neurexin
binding. In each case α-dystroglycan also showed a reduced molecular mass, likely due to
the massive reduction of sugar chains, and also did not bind to antibodies directed against
the sugar epitopes. These experiments strongly support the theory that α-dystroglycan
binds to ECM proteins as part of its function, and it is its glycans that are vital in successful
adhesion.
In the following sections I will cover a number of diseases involving mutations in various
confirmed and putative glycotransferases that when defective result in hypoglycosylation of
α-dystroglycan. Whilst individually each disease may cause different disease symptoms with
varying severity, they all share a secondary defect in dysfunctional α-dystroglycan protein.
Fukuyama-Type Congenital Muscular Dystrophy and Fukutin
Fukuyama-type congenital muscular dystrophy (FCMD) is a glycosylation related muscular
dystrophy and the 2nd most common muscle disease in japan (the 1st being Duchenne MD
6. 6
(Martin-Rendon & Blake, 2003)), with an average life expectancy between 10 – 20 years
(van Reeuwijk et al. 2004). It is also one of the three cobblestone lissencephalies, the other
two being Walker-Warburg syndrome and muscle-eye-brain disease. Patients with FCMD
show mental retardation and neuronal migration abnormalities, in addition to the usual MD
symptoms. Epilepsy and other eye abnormalities can also seen, albeit present in a much
milder form, if at all. This helps differentiate FCMD from the more severe muscle-eye-brain
disease and Walker-Warburg syndrome (van Reeuwijk et al. 2004). FCMD is most commonly
caused by the insertion of an ancient retrotransposon into the 3’ untranslated region (UTR)
of the FCMD gene, causing destabilization of the mRNA transcript (Kobayashi et al. 1998).
Since point mutations have only been described when in the presence of retrotransposon
insertion in compound heterozygotes, it can be assumed that true null mutations of FCMD
are either lethal or extremely rare (Kondo-Iida et al. 1999). This highlights the importance of
a functional Fukutin protein during embryonic development.
The FCMD gene produces the Fukutin protein. It contains the Asp-Xaa-Asp (DxD) (Xaa/X
being any amino acid except proline) motif in its C-terminus, a sequence conserved among
many glycosyltransferase family members and has been shown to be vital in their enzymatic
activity (Breton & Imberty, 1999). The motif appears to help coordinate divalent metal
cations, such as Mn2+, which are required for the binding of nucleoside sugar phosphates in
numerous glycosyltransferases (Tarbouriech et al. 2001) (Wiggins & Munro, 1998). Also in
common with numerous glycosyltransferases, fukutin is targeted to the Golgi apparatus via
a signal anchor sequence in its N-terminus (Kobayashi et al. 1998) (Esapa et al. 2002). Since
the DxD motif is present in enzymes other than glycosyltransferases, it can be assumed its
main function is that of glycan recognition. This is certainly the case in GM2 synthase, which
transfers glycans to a lipid backbone via its DxD motif.
Whilst the exact enzymatic function of Fukutin has yet to be elucidated, α-dystroglycan
proteins isolated from the muscle of FCMD patients are abnormally processed, supporting
Fukutin’s importance in α-dystroglycan glycosylation. Recently it has been found that
fukutin may play an indirect role in α-dystroglycan glycosylation via interaction with
POMGnT1, another glycosyltransferase which resides in the Golgi (Xiong et al. 2006).
POMGnT1 plays a vital role in the addition of an N-acetylglucosamine (GLcNAc) residue to
an O-mannose linked protein, and Xiong et al. found that fukutin interacts with POMGnT1
via the transmembrane region of fukutin. They observed an approximately 30% decrease in
POMGnT1 activity in fukutin-defective mouse brain tissue, suggesting a potential association
between fukutin and POMGnT1 in vivo. Additionally, it has been shown that overexpression
of POMGnT1 in FCMD cells cannot repair the hypoglycosylated α-dystroglycan phenotype,
thus supporting the hypothesis that POMGnT1 requires fukutin in order to function properly
(Barresi et al. 2004). From this, it is feasible to think that fukutin may possess some kind of
chaperone function, in that it indirectly helps to glycosylate α-dystroglycan via interaction
with other glycosyltransferases.
7. 7
Congenital Muscular Dystrophy type 1C, Limb Girdle Muscular Dystrophy 2I and
Fukutin-Related Protein
Both Congenital Muscular Dystrophy type 1C (MDC1C) and Limb Girdle Muscular Dystrophy
2I (LGMD2I) are caused by mutations of varying severity in the gene encoding fukutin-
related protein (FKRP). FKRP bears a close resemblance to Fukutin due to the fact that they
are distant homologues, and they both contain the DxD motif in their C-terminus. This
suggests it may have some glycosyltransferase activity (Aravind & Koonin, 1999) (Breton &
Imberty, 1999). Depending on the severity of the mutation, either MDC1C (the more severe
form) or LGMD2I (the milder form) can be produced. MDC1C is a rare disease characterised
by severe muscular dystrophy arising within the first few weeks of life. LGMD2I is the more
common form and is relatively less dangerous as the average age of onset lies between
childhood and adulthood. MDC1C and LGMD2I differ from other CMDs in that eye and brain
defects commonly seen are absent, instead only showing the muscular dystrophy
phenotype (Lin et al. 2007). It is for this reason that MDC1C and LGMD2I are not classed as
cobblestone lissencephalies. Recently however, certain mutations in the FKRP protein have
been shown to cause muscle-eye-brain disease and Walker-Warburg syndrome, both of
which show significant eye and brain abnormalities (Beltran-Velaro de Bernabe et al. 2004).
As with FCMD, two null alleles have not been described for FKRP, suggesting FKRP must play
a vital role in development. Worth noting is that if a patient has a compound heterozygous
mutation for L276I and a null allele, they are still able to present the milder phenotype of
LGMD2I, highlighting how only one copy of the L276I mutant allele is required to prevent
the severe phenotype from surfacing (Keramaris-Vrantsis et al. 2007). Unlike FCMD, FKRP
doesn’t tend to cluster in isolated populations, due to the relative frequency of the
mutations causing MDC1C and LGMD2I. The L276I mutation mentioned previously involves
a substitution with a conserved amino acid (Brockington et al. 2001). Even though this small
change appears to be harmless, such changes in the sequences of glycosyltransferases can
have massive impacts on their substrate specificity and enzyme kinetics (Seto et al. 1999). A
novel compound heterozygous mutation, c.948delC and c.823C > T (R275C), was recently
found in an Asian patient which gave rise to LGMD (Lin et al. 2007). At the time of publishing,
LGMD2I was most prevalent throughout Europe and Africa, making this the first case of
LGMD2I in Asia. This highlights how common mutations that cause MDC1C and LGMD2I can
arise.
Whilst the exact function of FKRP remains unknown, heterologous overexpression of FKRP
in CHO cells shows a relative increase in protein glycosylation, resulting in an abundance of
immature, partially processed α-dystroglycan in vitro (Esapa et al. 2002). During protein
maturation, glycans on proteins require trimming down to specification, and due to the
overexpression of FKRP in these CHO cells, this maturation process could be disturbed by
the increased amount of glycan addition to the sugar chains which are never trimmed.
Additionally, when FKRP is mutated, for example in MDC1C and LGMD2I, α-dystroglycan
8. 8
glycosylation activity is abolished or greatly inhibited, indicating FKRP performs some vital
role in the glycosylation of α-dystroglycan. Also worth considering is the new found
possibility that fukutin may serve a chaperone like function. Since FKRP and fukutin have
very similar sequence homology, FKRP may too serve to assist protein glycosylation but not
partake in it directly.
Similar to Fukutin, FKRP resides in the Golgi apparatus and is targeted using a signal anchor
sequence within its N-terminus (Esapa et al. 2002). Using Chinese hamster ovary (CHO) cells,
it was shown that the P448L mutation prevents FKRP entering the Golgi apparatus (Esapa et
al. 2002), causing the MDC1C phenotype. Since no α-dystroglycan processing was seen, it
can be assumed that targeting to the Golgi is essential for the function of FKRP. It has been
shown (Esapa et al. 2005) that the most severe FKRP mutations which cause CMDs,
including those with and without brain defects, cause retention of the FKRP protein within
the ER where it has a shorted half-life and is preferentially degraded by proteasomes.
However, in the L276I mutation FKRPs are still able to be trafficked to the Golgi apparatus
where they have much greater protection from proteasomal degradation. This is mirrored
by wild-type FKRP, which is also transported to the Golgi. This strongly supports the theory
that FKRP requires localization within the Golgi apparatus in order to properly function. It is
possible that the differences in disease severity between CMDs with brain defects, MDC1C
and LGMD2I are caused by differential trafficking of the mutated FKRP within the cell. Each
of the mutants may retain some function but can’t act upon their specific substrates as they
cannot reach the Golgi apparatus (Esapa et al. 2002). This is clearly shown in the LGMD2I
causing L276I mutant, where patients show almost normal α-dystroglycan processing, likely
due to the fact that FKRP is able to be localized within the Golgi. Once the exact function of
FKRP has been elucidated, it will be interesting to see the how much activity the severe
mutants can retain when forcibly localized within the Golgi, potentially opening up
numerous treatment options.
Muscle Eye-Brain Disease and POMGnT1
As with FCMD, Muscle Eye-Brain (MEB) disease is a cobblestone lissencephaly found mostly
genetically isolated populations, except instead of Japan it’s the Finns (Cormand et al. 1999).
MEB has similar symptoms to FCMD, including mental retardation, neuronal-migration
disorders, cerebellar hypoplasia, and flattening of the brain stem. In addition, MEB presents
several severe eye disorders such as congenital myopia, congenital glaucoma, pallor of the
optic discs, and retinal hypoplasia (Xiong et al. 2006). The progression of MEB is much
slower than FCMD, so survival into adulthood is quite common, with life expectancy
between 10 – 30 years (van Reeuwijk et al. 2004). MEB is caused by a mutation in the O-
mannose b-1, 2-N-acetylglucosaminyl transferase (POMGnT1) gene (Yoshida et al. 2001).
POMGnT1 works by catalysing the addition of a GLcNAc residue to a peptide-linked
mannose residue. Like Fukutin and FKRP, POMGnT1 has an N-terminal transmembrane
domain and may be localized to the Golgi apparatus. However, instead of a DxD motif it has
9. 9
a Glu-Asp-Asp (EDD) motif. When POMGnT1 is mutated, abnormal processing is observed
on α-dystroglycan in MEB patients (Kano et al. 2002).
The majority of mutations identified in MEB disease are nonsense mutations which
prematurely terminate the reading frame (Yoshida et al. 2001). Missense mutations and
mutations caused by exon skipping events only showed catalytic activity <1% of the wild
type POMGnT1.
Walker-Warburg Syndrome and POMT1/POMT2
Walker-Warburg syndrome (WWS) is the last of the cobblestone lissencephalies, and causes
severe muscular dystrophy with patients possessing very little motor activity. Also
associated with WWS are various neuronal-migration defects, similar to FCMD and MEB, but
WWS patients also suffer from Agyria, Encephalocele and the absence of corpus callosum,
cerebellar vermis and septum (van Reeuwijk et al. 2004). WWS patients also suffer from
various other structural abnormalities of the brain and eye. WWS is most commonly caused
by mutations in protein O-mannosyltransferase 1(POMT1) gene (Beltran-Valero De Bernabe
et al. 2002) (Jurado et al. 1999), which catalyses the addition of a dolichyl phosphate
activated mannose residue directly to the polypeptide backbone. Because POMT1 serves to
initiate glycosylation on α-dystroglycan, any defects in these proteins are naturally going to
make the worst impact on α-dystroglycan processing. It is for this reason why WWS is the
most severe CMD and cobblestone lissencephaly of its kind, with patients having a life
expectancy of only 3 years (van Reeuwijk et al. 2004).
Unlike Fukutin, FKRP and POMGnT1, POMT1 does not have transmembrane domain in its N-
terminus; instead it has three transmembrane segments in its C-terminus. POMT1 also lacks
the common DxD motifs seen in Fukutin and FKRP, in addition to the EDD motif seen in
POMGnT1, highlighting its uniqueness from other glycosyltransferases. POMT1 belongs to
the family of protein mannosyltransferases (Pmts). These Pmts in yeast genomes appear to
be highly selective for their protein substrates, such that similar sugar modifications on
unrelated proteins are catalysed by different Pmt isoforms (Gentzsch & Tanner, 1996). From
this, it can be assumed that POMT1 must only catalyse the addition of mannose to a few
substrate types, of which includes α-dystroglycan. Since POMT1 and Pmts in general add
mannose directly to the polypeptide backbone, they are the first steps in O-linked protein
glycosylation. In agreement with this, Pmts functioning in the ER of yeast which fail to
properly add mannose will prevent subsequent modifications in the Golgi, leading to heavily
hypoglycosylated protein products, as is the case in WWS found in humans.
In humans and Drosophila flies there are two Pmt orthologues: POMT1 and POMT2.
Drosophila lacking in either functional POMT1 or POMT2 suffer from the rotated abdomen
(rt) phenotype, which, as the name suggests, causes rotation of the fly abdomen by 30-60°
(van Reeuwijk et al. 2005). This defect stems from improper muscle development in the fly,
being similar in nature to the muscular dystrophies seen in humans. Since both functional
10. 10
POMT1 and POMT2 are required for proper muscle development in the fly, Reeuwijk et al.
sought to find if this was the case in humans as well. They found three patients with
homozygous mutations in POMT2 who displayed WWS phenotypes indistinguishable from
those suffering from POMT1, FCMD, and FKRP mutations. Two mutations led to premature
stop codons and one was a splice site mutation. In their study they found the incidence of
POMT2 mutations matched those of POMT1, which agrees with the hypothesis that both
POMT1 and POMT2 together are required for O-mannosyltransferase activity, as described
in Drosophila above.
Largemyd Mouse and LARGE
Largemyd is an autosomal recessive muscular dystrophy found in mice (Lane et al. 1976).
Similar to the previous human disorders, Largemyd mice show the typical muscular dystrophy
syndromes along with neuronal-migration problems and abnormal retinal electrophysiology
(Holzfeind et al. 2002) (Michele et al. 2002). The LARGE protein is mutated in Largemyd mice
(Grewal et al. 2001) (Peyrard et al. 1999) and is a putative glycosyltransferase as it has two
Asp-Xaa-Asp motifs, raising the possibility that LARGE is bifunctional. LARGE also has two
coiled-coil regions flanking either side of the glycosyl-transferase family 8 (GT8) domain,
which may be sites for protein-protein interaction (Blake et al. 1995). Largemyd mice show
altered α-dystroglycan glycosylation, confirming its relation to muscular dystrophies and the
DAPC in general (Grewal et al. 2001).
Recent work has found that overexpression of endogenous LARGE in Largemyd mouse
restores the levels of properly glycosylated α-dystroglycan and reduces the muscular
dystrophy phenotype (van Reeuwijk et al. 2004). Perhaps more interestingly, overexpression
of LARGE in cells taken from patients suffering from FCMD, MEB and WWS showed an
increase in correctly glycosylated α-dystroglycan, indicating a possible regulatory role of
LARGE in the glycosylation of α-dystroglycan (Barresi et al. 2004), similar to fukutin (Xiong et
al. 2006). In agreement with this, it has been shown that LARGE does indeed play a role in
mediating the O-linked glycosylation α-dystroglycan via molecular interaction with the N-
terminal domain of α-dystroglycan (Kanagawa et al. 2004). How LARGE is able to
compensate for the reduced functionality of mutant FCMD, MEB and WWS, however, has
yet to be elucidated.
Genotype-Phenotype Correlations
Linking the genotypic properties of the various glycosyltransferases involved and the clinical
severity of the diseases they can produce when mutated is a fairly complex task. Numerous
factors are involved; such the functional step a glycosyltransferase partakes in during O-
linked glycosylation, and the severity of mutations affecting the glycosyltransferases (van
Reeuwijk et al. 2004).
11. 11
POMT1/2 is a great example of how a glycosyltransferases role correlates with its associated
disease phenotype. POMT1/2 action is predicted to be the first catalytic step in O-linked
protein glycosylation; through the transfer of a mannose residue from dolichyl phosphate
mannose to either a serine or threonine residue to the target protein (Jurado et al. 1999). If
this highly important function is defective, then the subsequent processing of target
proteins is going to be significantly inhibited, resulting in a more hypoglycosylated protein.
The hypoglycosylation caused by mutants in POMT1/2 is going to be more severe than those
caused by mutants in, say, POMGnT1, which catalyses the addition of a GlcNAc to the
mannose residue already added by POMT1/2. It is for this reason that WWS, the disease
most often caused by POMT1 mutations, is the most potent CMD of its type, presenting a
strong genotype-phenotype correlation between glycosyltransferase function and disease.
Mutations in glycosyltransferases that normally produce one disease phenotype also have
the potential to produce other, more severe diseases. For example, WWS is normally caused
by POMT1/2 mutations, but it can also be caused by mutations in the fukutin and FKRP
genes (van Reeuwijk et al. 2004). The comparatively mild FCMD is caused by a
retrotransposon insertion into the 3’ UTR, which although reduces the level of Fukutin
mRNA, said mRNA is still able to produce functional Fukutin (Kondo-Iida et al. 1999).
However, when the fukutin gene undergoes much more severe homozygous nonsense
mutations, the WWS phenotype is produced due to the resulting heavily dysfunctional
fukutin protein. This indicates some genotype-phenotype correlation between the severity
of the mutation and the severity of the disease produced; mild mutations tend to produce
milder disease phenotypes, such as FCMD, and more damaging mutations tend to produce
more severe disease phenotypes, such as WWS.
FKRP gene mutations provide a somewhat less clear example of genotype-phenotype
correlations in the CMDs. FKRP gene mutants are known to mostly cause LGMD2I and
MDC1C, but can also be found in MEB and WWS as well. Owing to the fact we don’t know
what FKRP actually does, outside of it being a putative glycosyltransferase, it’s hard to
establish solid genotype-phenotype correlations (van Reeuwijk et al. 2004). Since most FKRP
mutations are amino acid substitutions, it’s hard to pin point their exact impact on FKRP due
to our lack of knowledge to its function. It is still possible to draw some correlations,
however. As mentioned previously, certain FKRP mutations which cause retention of novel
FKRP proteins within the ER are mainly associated with the more severe MDC1C, and those
which permit translocation of FKRP to the Golgi result the milder LGMD2I. Whilst we do not
know why certain mutations cause retention of FKRP in the ER, we can still deduce that a
genotype-phenotype correlation exists between the localisation of FKRP and the severity of
disease (Esapa et al. 2002).
POMGnT1 mutations also provide an example of genotype-phenotype correlations.
POMGnT1 is mostly associated with MEB in the Finnish population, similar to FCMD in Japan,
but recently numerous POMGnT1 gene mutations have been associated with a number of
12. 12
different disease phenotypes identified in patients all over the world (van Reeuwijk et al.
2004). A genotype-phenotype trend was observed in the location of mutations affecting the
POMGnT1 gene, where mutations nearer to the 5’-terminus of the POMGnT1 coding region
produced more severe mutations. These mutations caused disease phenotypes similar to
but not as damaging as WWS, with brain defects such as hydrocephalus. Towards the 3’-
terminus mutations tended to cause less severe disease phenotypes with an absence of
hydrocephalus, suggesting mutations closer to the 5’-terminus of the POMGnT1 gene make
a much greater impact than those further away. The molecular basis for this could be due to
disruption of POMGnT1 targeting, as the transmembrane signal anchor sequence within the
POMGnT1 gene lies close to the 5’-terminus, which when disrupted by mutations may cause
mislocalisation of POMGnT1, similar to FKRP mislocalisation. Mutations in the EDD domain
would be comparatively less damaging as POMGnT1 would still be able to perform limited
catalytic activity, assuming the mutation itself wasn’t too severe, whereas mislocalised
mutants wouldn’t be able to reach their specific substrates at all and therefore be
considerably less functional.
Conclusion
Over the past few years, great progress has been made concerning the dystroglycan
glycosylation pathway and the associated dystroglycanopathies. The correct glycosylation of
α-dystroglycan is vital to its function, and mutations affecting glycosyltransferases specific
for α-dystroglycan result in many different types of CMD. The most severe, WWS, is caused
by mutations in POMT1/2, which catalyse the addition of mannose directly to the peptide
backbone, are both required in order for either protein to function correctly. MEB, being
somewhat less severe than WWS, is caused by mutations in POMGnT1 which adds a GlcNAc
residue to an O-mannose linked protein. Then there’s FCMD, caused by fukutin, which is
largely less damaging than both MEB and WWS. FKRP can cause both the mild LGMD2I and
the severe MDC1C, depending on the nature of mutations affecting it. However, things such
as the exact function of fukutin and FKRP and how the large majority of genes accounting
for WWS remain unknown show how there is still a lot yet to be found out. Also the way
LARGE is able to restore dystroglycan glycosylation in MEB, WWS and FCMD remains to be
seen.
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