Biosci. Biotechnol. Biochem.

, 71 (8), 1811–1818, 2007

Award Review
Mammalian Glycerophosphodiester Phosphodiesterases
Noriyuki Y ANAKA
Department of Molecular and Applied Bioscience, Graduate School of Biosphere Science,
Hiroshima University, 4-4, Kagamiyama 1-chome, Higashi-Hiroshima 739-8528, Japan

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Online Publication, August 7, 2007
[doi:10.1271/bbb.70062]

Bacterial glycerophosphodiester phosphodiesterases O O

(GP-PDEs), GlpQ and UgpQ, are well-characterized
periplasmic and cytosolic proteins that play critical
roles in the hydrolysis of deacylated glycerophospholi-
pids to glycerol phosphate and alcohol, which are
utilized as major sources of carbon and phosphate. In
contrast, two novel mammalian GP-PDEs, GDE1/
MIR16 and GDE3, were recently identified, and were
shown to be involved in several physiological functions.
GDE1/MIR16 was identified as a membrane protein
interacting with RGS16, a regulator of G protein
signaling, and found to hydrolyze glycerophosphoinosi-
tol preferentially. We have found that expression of
GDE3 is significantly up-regulated during osteoblast
differentiation and is involved in morphological changes
of cells. Furthermore, five mammalian GP-PDEs were
virtually identified, and very recent studies indicate that
retinoic acid-induced expression of GDE2 plays essen-
tial roles in neuronal differentiation and neurite out-
growth. Thus mammalian GP-PDEs are likely to be
important in controlling numerous cellular events,
indicating that the GP-PDE superfamily in mammals
might be a pharmacological target in the future.
Key words: glycerophosphodiester phosphodiesterases;
glycerophosphoinositol; GDE; GlpQ
The Escherichia coli (E. coli) glycerophosphodiester
phosphodiesterases (GP-PDEs) GlpQ and UgpQ are well
known as periplasmic and cytosolic proteins with
important functions in the uptake of sn-glycerol-3-
phosphate.1,2) Bacterial GP-PDEs catalyze the hydroly-
sis of deacylated glycerophospholipids to glycerol
phosphate (Fig. 1), which is a major source of carbon
and phosphate. This reaction also generates correspond-
ing alcohols, such as choline and ethanolamine, that act
as essential growth factors for bacteria. In contrast,
Haemophilus influenzae (H. influenzae) GlpQ, an outer
membrane protein, was originally isolated as a protein
Address correspondence to; Tel: +81-82-424-7979; Fax: +81-82-422-7067; E-mail: yanaka@hiroshima-u.ac.jp
Abbreviations: GP-PDE, glycerophosphodiester phosphodiesterase; RGS, regulator of G protein signaling; GroPIns, glycerophosphoinsitol;
GroPIns-4P, glycerophosphoinsitol 4-phosphate; EST, expressed sequence tags; GFP, green fluorescent protein
This review was written in response to the author’s receipt of The Japan Bioscience, Biotechnology, and Agrochemistry Society Award for the
Encouragement of Young Scientists in 2005.
R O P O CH2 O P O CH2
ROH
O CHOH O CHOH
CH2OH CH2OH
glycerophosphodiester phosphodiesterase
Fig. 1. Enzymatic Reaction of Bacterial Glycerophosphodiester
Phosphodiesterases.
Bacterial GP-PDEs hydrolyze deacylated phospholipids glycer-
ophosphodiesters, such as glycerophosphocholine and glycerophos-
phoethanolamine, to sn-glycerol-3-phosphate and corresponding
alcohols.
responsible for interaction with immunoglobulin D and
was found to have GP-PDE activity.3–5) In a rat otitis
model, a GlpQ mutant required an approximately 100
fold-higher inoculum than the parental H. influenzae in
order to induce otitis media after injection, strongly
suggesting that GlpQ plays an important role as a
virulence factor in H. influenzae.6,7) Recently, a novel
physiological role of H. influenzae GlpQ, closely related
to the adherence of bacteria to human cells, was
proposed. H. influenzae incorporates choline obtained
from abundant pools of degradation products of the host
cell membrane lipid by using GlpQ in its lipopolysac-
caride as phosphorylcholine, which contributes to the
pathogenesis by mimicry of the host cell membrane.6)
E. coli GlpQ is a periplasmic protein, whereas this
enzyme of H. influenzae is a lipoprotein located in the
outer membrane, suggesting that the difference in the
cellular location of these bacterial GP-PDEs might
provide functional diversity among these enzymes.
In mammals, two GP-PDEs, GDE1/MIR16 and
GDE3, have been independently identified. Zheng et
al. used a yeast two-hybrid system to define the RGS16
(for regulator of G protein signaling) mediated signaling
pathway, and isolated GDE1, a membrane protein
interacting with RGS16.8) In contrast, we employed a
1812 N. YANAKA

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Fig. 2. The Deduced Amino Acid Sequences of the Catalytic Region of Mouse GP-PDEs.
The deduced amino acid sequences of the putative catalytic region of mouse GDE1-7 proteins are aligned for the best match. Identical amino
acid residues are boxed.

GDE
GDE1 N- -C

GDE
GDE2 N- -C

GDE
GDE3 N- -C

GDE
GDE4 N- -C
Carbohydrate binding
domain GDE
GDE5 N- -C

GDE
GDE6 N- -C

GDE
GDE7 N- -C

Fig. 3. Schematic Diagrams Illustrating Domain Structures of the Seven Mouse GP-PDEs.
Putative transmembrane domains are numbered from the N-terminal. GDE represents the putative GP-PDE domain.

differential display method to isolate the components I. Identification of GDE1/MIR16 as a Mem-

involved in osteoblast differentiation, and isolated
GDE3, which is specifically expressed at the stage of
matrix maturation.9) Although these studies indicated
that mammalian GP-PDEs are involved in numerous
physiological functions, including signal transduction
and cytoskeletal regulation, probably many GP-PDE
homologs remain unidentified in mammals and are
likely to play distinct physiological roles. Because GP-
PDE proteins contain a highly conserved region des-
ignated the GDE domain that comprises approximately
56 amino acid residues (Fig. 2), a search of databases of
expressed sequence tags (ESTs) based on their amino
acid sequence homology is expected to be an effective
approach to the isolation of novel GP-PDE cDNAs.
To date, seven mammalian GP-PDEs have been vir-
tually cloned by this approach using bioinformatics
(Fig. 3).10,11) Characterization of these GP-PDEs will be
very important in order to understand complex mech-
anisms controlling phospholipid metabolism and to find
pharmacologically significant molecules. In this review,
I will introduce the current knowledge concerning
mammalian GP-PDEs.
brane Protein Interacting with RGS16
Heterotrimeric G-proteins are known for their trans-
duction of extracellular signals from G protein-coupled
receptors to downstream G protein effectors.12) RGS
proteins, regulators of G-protein signaling, belong to a
large gene family and include four members, which
regulate the G-protein-mediated signaling pathway by
acting as GTPase activating proteins.13,14) To date, 24
RGS proteins have been identified in mammals, and they
are thought to play distinct physiological roles. In
particular, Farquhar et al. are interested in the G-protein-
mediated signaling pathway associated with the intra-
cellular membrane that is involved in the secretory
system.15,16) They focused on RGS1617) one of the RGS
proteins, which was highly expressed in the mouse
pituitary and liver, and used a yeast two-hybrid system
using RGS16 as a bait to define the signaling pathway
mediated by RGS16. They identified a cDNA that
encodes a novel protein (named GDE1) composed of
311 amino acids with a putative glycerophosphodiester
phosphodiesterase region, and characterized the inter-
 Mammalian Glycerophosphodiester Phosphodiesterases
action with RGS16 and the expression of GDE1 in
detail.8,10) Northern blot analysis revealed that GDE1 is
widely expressed in mammalian tissues, including the
heart, brain, liver, and kidney. Analyses of the hydro-
phobicity profiles of GDE1 protein indicated that two
distinct hydrophobic regions are located at the N-
terminus and at the C-terminus. Two hydrophobic
regions were also found in GDE4 and GDE7 proteins
as well as in bacterial GP-PDEs. To investigate the
membrane topology of GDE1 further, they employed
proteinase K protection assays, showing that a catalytic
domain between two transmembrane domains faces
either the lumen of the endoplasmic reticulum (ER) or
the extracellular space.10) In their study, GDE1 protein
was also shown to be localized mainly on the plasma
membrane in the liver and kidney, and in the juxtanu-
clear region of cultured pituitary cells and PC-12 cells.
Of much interest to us is the fact that expression of wild-
type GDE1 in HEK293T cells led to a significant
increase in glycerophosphoinsitol (GroPIns) phospho-
diesterase activity.10) Because bacterial GP-PDEs are
well characterized as exhibiting broad catalytic activity
and for hydrolyzing glycerophosphocholine, glycero-
phosphoserine, glycerophosphoethanolamine, glycero-
phosphoglycerol, bis (glycerophospho)-glycerol, and
GroPIns, it is intriguing that GDE1 was found to
hydrolyze GroPIns preferentially, with an apparent Km
of 12 mM and a Vmax of 3;000 pmol/mg/min. A
substrate competition assay showed that the competitive
preference for GDE1 was GroPIns 4,5-bisphosphate 
glycerophosphoserine > GropIns > GroPIns 4-phos-
phate (GroPIns-4P)  glycerophosphoethanolamine  GDE3 was also expressed in the spleen as well as in
glycerophosphocholine. It is noteworthy that isoproter-
enol, an agonist for
-adrenergic receptors, increases
GDE1 phosphodiesterase activity toward GroPIns by
50% as compared with untreated cells. In contrast,
phenylephrine, an agonist for -adrenergic receptors,
and lysophosphatidic acid reduced the phosphodiester-
ase activity by 30 and 40% respectively. Although these
interesting observations by Zheng et al.10) strongly
suggest that the GroPIns phosphodiesterase activity of
GDE1 is regulated by G protein signaling pathways, it
remains to be determined whether the interaction of
GDE1 with RGS protein regulates GroPIns activity.
Very recently, Bachmann et al. proposed a molecular
3D model of the catalytic region of human GDE1 by
comparing the amino acid sequences between mamma-
lian GDE1 and bacterial GP-PDE proteins.18) Based on
the structural model, the catalytic region of GDE1
protein consists of a central
-sheet made up of eight
strands, two additional
-strands, and 13 -helices. The
proposed model suggests that the predicted a/
structure
resembles a triose-phosphate-isomerase (TIM) barrel
domain, and that the conserved core amino residues are
essential for GDE1 catalytic activity, providing impor-
tant information for an understanding of the molecular
function of mammalian GP-PDE proteins.
1813
II. GDE3 Promoted Osteoblast Differentia-
tion and Induced Morphological Changes in
Cells
Osteoblasts are mesenchymal cells derived from bone
marrow stromal cells, periosteum, or undifferentiated
stem cells in connective tissues.19,20) The process of
osteoblast differentiation has been well characterized at
the morphological level. The process can be subdivided
into three main phases: proliferation, extracellular
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matrix development, and mineralization. Numerous
investigations have been directed at elucidating the
regulation of bone formation, and have determined that
many genes encoding either transcription factors or
growth factors are essential for osteoblast differentia-
tion.21,22) MC3T3-E1 cells are a well-established pre-
osteoblast cell line derived from mouse calvaria, and are
widely used for studies of osteoblast differentiation
induced by
-glycerophosphate and ascorbic acid. In
order to identify new osteoblast-related genes, we
cloned cDNAs that were differentially expressed during
specific differentiation stages in mouse osteoblast-like
MC3T3-E1 cells by a differential display method. We
isolated a novel cDNA encoding a putative glycero-
phosphodiester phosphodiesterase, GDE3, which is
transiently expressed at the stage of matrix maturation
(Fig. 4A).9) The deduced amino acid sequence predicted
that GDE3 contains 539 amino acids, including seven
putative transmembrane domains and a putative glycer-
ophosphodiester phosphodiesterase region in a large
extracellular loop. Northern blot analysis revealed that
primary calvarial osteoblasts and the femur. We ob-
served that green fluorescent protein (GFP)-fused GDE3
protein accumulates at the cell periphery in transfected
HEK293T cells, and that the transfected cells change
from a spread form to a rounded form (Fig. 4B) with the
disappearance of actin filaments.9) Immunofluorescence
staining with GDE3 antibody in MC3T3-E1 cells
suggested that endogenous GDE3 is co-localized with
the actin cytoskeleton. To determine the physiological
role of GDE3 in osteoblast differentiation, MC3T3-E1
cells stably expressing GDE3 were constructed. Stable
expression of GDE3 was found to lead to dramatic
increases in alkaline phosphatase activity and calcium
deposits. In this study, we prepared anti-GDE3 antibody
specifically recognizing the putative GP-PDE domain in
the extracellular loop using a bacterially synthesized
maltose-binding protein-fused fragment corresponding
to amino acids 219–332 of GDE3. Because FACS
analysis using purified anti-GDE3 antibody indicated
that the putative GP-PDE domain faces an extracellular
space like GDE1, we are interested to know whether this
antibody has a neutralizing effect on the physiological
functions of GDE3 during the differentiation of MC3T3-
E1 cells. The addition of anti-GDE3 antibody to the
culture medium caused a 72.5% decrease in alkaline
phosphatase activity as compared to that of normal
1814 N. YANAKA
A nervous system development, including neuronal differ-
0 4 7 11 15 (days)
GDE3
Osteopontin
Osteocalcin
TypeI collagen
B remains limited. In a very recent study, Rao et al.
GFP
GDE3-GFP
Fig. 4. GDE3 Is Involved in Morphological Changes in Cells and
Promotes Osteoblast Differentiation.
A, GDE3 mRNA increased transiently during differentiation of
MC3T3-E1 cells. Total RNA from MC3T3-E1 cells cultured at the
indicated days was subjected to Northern blot analysis. Filters were
probed for GDE3, osteopontin, osteocalcin, and type I collagen. B,
HEK293T cells were transiently transfected with green fluorescence
protein (GFP) or GFP-fused GDE3 (GDE3-GFP). The expression of
GDE3 induced cell rounding (GDE3-GFP, left panel). Wild-type
GFP was distributed evenly over the entire cytoplasm in HEK293T
cells (GFP, right panel), whereas GDE3 protein was concentrated at
the cell periphery (GDE3-GFP, right panel).
rabbit IgG.9) Taken together with these observations, it
is probable that GDE3 accelerates the program of
osteoblast differentiation of MC3T3-E1 cells.
III. Virtual Cloning of GDE2 and It’s
Physiological Roles in Neuronal Differentia-
tion
The precise wiring of neural circuits and connections
formed between neurons depends on the ability of
developing neurons to extend axons and dendrites to
their targets.23,24) Neurite outgrowth is regulated by a
variety of signaling mechanisms, including growth
factors and soluble or membrane-bound guidance cues,
with coordinated regulation of the cytoskeleton within
growth cones, which exhibit highly motile actin-based
structures.25) Recent studies have indicated that the Rho
family of small G-proteins, Rho, Rac, and Cdc42, are
key proteins in the regulation of trasnsducing signals
from extracellular cues to the actin cytoskeleton.26,27) On
the other hand, retinoic acid (RA), an active metabolite
of vitamin A, is known to play important roles in
entiation, patterning, and outgrowth.28) In particular, RA
is known to induce neurite outgrowth from the spinal
cord, dorsal root ganglia neurons, and neuroblastoma
cells.29–31) Previous reports have demonstrated that one
mechanism whereby RA regulates neuronal outgrowth is
by modulation of the expression of neurotrophin
receptors, including TrkA and p75NTR ,30,32) but our
understanding of the mechanisms involved in the control
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of neuronal differentiation and outgrowth by RA
cloned cDNAs that differentially expressed in ventral
spinal cord explants grown in the presence or absence of
RA by a differential subtractive screen, and identified
GDE2 as a new RA-responsive gene in motor neu-
rons.33) Motor neuron progenitors located in the ven-
tricular zone of the spinal cord highly express oligoden-
drocyte transcription factor 2 (Olig2). These progenitor
cells exit the cell-cycle, migrate laterally, and begin to
express motor neuron-specific transcription factors,
including homeobox factor 9 (HB9), islet1, and islet2.
It was found that GDE2 mRNA is highly expressed in
mature motor neurons that are postmitotic and express
HB9, islet1, and islet2 in the chick spinal cord. In ovo
electroporation of GDE2 small interfering RNAs
(siRNAs) resulted in a 70% loss of HB9-espressing
neurons and a 30 to 40% loss of more mature motor
neurons expressing islet2.33) Furthermore, Rao et al.
found that expression of the motor neuron markers HB9
and islet2 is induced in progenitor cells expressing Olig-
2 in response to electroporation of GDE2 cDNA. In
contrast, conversion of a conserved histidine residue to
alanine in the GP-PDE region of GDE2 abolished the
ability of GDE2 to promote motor neuron differentia-
tion.33) These interesting observations by Rao et al.
strongly suggest that GDE2 is primarily expressed in
mature neurons and that it plays important roles in motor
neuron differentiation through GP-PDE activity.
As described above, RA is known to induce neurite
outgrowth of human and mouse neuroblastoma cells.
Ever since we showed that GDE3 protein is localized at
the cell periphery and plays a critical role in morpho-
logical changes in cells, as described above, we have
been particularly interested in the physiological func-
tions of mammalian GP-PDEs during neuronal out-
growth/retraction. In order to isolate GP-PDEs that are
highly expressed in neuronal tissues, a search of EST
databases was performed using their putative catalytic
domains. We found that GDE2 mRNA was highly
expressed in a variety of brain regions,34) including the
cerebral cortex, cerebellum, hippocampus, and primary
cortical neurons. Because these observations including
novel functions of GDE3 led us to hypothesize that
GDE2 is involved in neuronal outgrowth, we inves-
tigated the physiological function of GDE2 during
neuronal differentiation by RA in Neuro2A cells, which
are neuroblastoma from the mouse spinal cord and are
 Mammalian Glycerophosphodiester Phosphodiesterases 1815

A
Retinoic acid
0 1 3 6 14 26 hrs

GDE2

-actin

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B
Scramble GDE2 RNAi

Fig. 5. GDE2 Is Involved in Neuronal Outgrowth.

A, Differentiation and neuritogenesis of Neuro2A cells were induced by administration of retinoic acid. Total RNA from Neuro2A cells
cultured at the indicated times was subjected to Northern blot analysis. B, Effect of GDE2 siRNA on neurite formation of Neuro2A cells.
Neuro2A cells were transfected with scrambled siRNA (scramble) or GDE2 siRNA (GDE2 RNAi) and then stimulated with retinoic acid (RA).
The morphology of these cells, cultured in the presence of RA for 48 h, was observed under a microscope, and photographs were taken at 100
magnification.

widely used in studies of neuronal outgrowth. Our first
finding was that expression of GDE2 mRNA in
Neuro2A cells was significantly up-regulated during
neuronal differentiation by RA treatment (Fig. 5A).34)
To identify the role of GDE2 in neuronal differentiation,
Neuro2A cells stably expressing the full-length protein
were constructed. We found that expression of GDE2
resulted in significant increases in neurite length and
numbers of cells bearing neurites in the absence of RA.
Furthermore, we observed that GFP-fused GDE2 accu-
mulates in the regions of perinuclear and growth cones
in Neuro2A cells and that a loss of function of GDE2 in
Neuro2A cells by RNAi blocks RA-induced neurite
formation (Fig. 5B).34) These observations indicate that
GDE2 expression during neuronal differentiation plays
an important role in growing neurites.
IV. Physiological Functions of GP-PDEs
GDE1 was recently found to hydrolyze GroPIns
preferentially.10) Endogenous GroPIns and its phos-
phorylated form, GroPIns-4P, are intracellular, water-
soluble metabolites, and were found to be elevated under
experimental conditions of Ras-transformation in a
number of cell lines.35,36) In addition, intracellular
accumulation of these compounds has been reported to
be correlated to the differentiation/dedifferentiation of
several cell lines, including macrophages, hapatocytes,
and neuronal cells.37,38) GroPIns and GroPIns-4P have
been found to modulate various cellular events, such as
intracellular cyclic AMP production and invasion of the
extracellular matrix by tumor cells.39,40) Mancini et al.
recently identified remarkable effects of GroPIns-4P in
the reorganization of the actin cytoskeleton, showing
that exogenously added GroPIns-4P rapidly and potently
induced formation of membrane ruffles and stress fibers
in Swiss 3T3 cells.41) We found that endogenous GDE3
protein appears to be co-localized with the actin
cytoskeleton in MC3T3-E1 cells, and that overexpres-
sion of GDE3 changed transfected HEK293T cells from
a spread to a rounded form. Taken together with these
observations, it is suggested that mammalian GP-PDEs
function in actin cytoskeletal organization as a rate-
limiting enzyme that is a critical determinant of intra-
cellular GroPIns and GroPIns-4P levels. An obvious
question that arises from these results is where GroPIns
is degraded, because the catalytic domains of mamma-
lian GP-PDEs, except for GDE5, face either the lumen
of the endoplasmic reticulum or the extracellular space.
GroPIns has been found to be present in both intra-
cellular and extracellular spaces.37) Exogenously added
GroPIns is able to cross the plasma membrane to reach
equilibrium rapidly with the cell cytosol and exert its
biological actions intracellularly, implying the existence
of a GroPIns transporter in mammalian cells. In yeast,
GroPIns is known to be transported across the plasma
1816
membrane by a permease encoded by the GIT1
gene.42,43) Very recently, Mariggiò et al. have found
that Glut2 transporter is a human-genome candidate
ortholog GIT1, and that it functions as a GroPIns
transporter in mammals.44) The existence of a GroPIns
transporter in mammalian cells suggests that the intra-
cellular concentration of GroPIns might be regulated by
GP-PDE activity in the extracellular space.
On the other hand, we recently found that GDE2 is
involved in the neuronal outgrowth of Neuro2A cells.34)
It has been proposed that an increase in cell membrane
surface area is also necessary during neuronal out-
growth.45) A continuous supply of newly synthesized
membrane components is necessary to sustain growth.
Previous reports have demonstrated that arachidonic
acid-releasing phospholipases (PLA2) are highly en-
riched in growth cones, and that exogenously added
PLA2 can promote neurite extension.46,47) Very recently,
Darios et al. reported that arachidonic acid stimulated
cell membrane expansion by interacting with syntaxin 3,
a plasma membrane protein.48) These observations
suggest that soluble fatty acids such as arachidonic acid
released from the plasma membrane play an important
role in neuronal cell membrane expansion. GroPIns is a
deacylation product of the membrane phosphatidylino-
sitol formed by sequential actions of PLA1/PLA2 and
lysophospholipases.37) After GP-PDEs hydrolyze Gro-
PIns to glycerol phosphate and inositol, these final
catabolic products are most likely utilized in phospho-
lipid synthesis as a recycling pathway, one that might be
essential for the constitutive release/supply of soluble
fatty acids such as arachidonic acid during neuronal
formation.
V. Other Mammalian GP-PDEs
As described in this review, mammalian GP-PDEs are
thought to be involved in numerous physiological
functions, including cytoskeletal regulation, signal trans-
duction, and cell differentiation. To identify a novel GP-
PDE, a search of databases of ESTs was carried out
using parts of the mammalian GP-PDE sequences. This
approach has been found to be effective in the isolation
of novel GP-PDE cDNAs. Recently, we isolated a novel
cDNA encoding a serpentine membrane protein, GDE6,
containing a GP-PDE motif from mouse cDNA libra-
ries.11) The deduced sequence of GDE6 was composed
of 633 amino acids with seven putative transmembrane
regions. As shown in Fig. 3, an intriguing feature of
GDE6 is that the GP-PDE domain is found within
transmembrane domains VI and VII. The predominant
signals of GFP-tagged GDE6 protein were found to be
localized primarily in the perinuclear region. Because
GDE6 transcript is known to be particularly abundant in
the spermatocytes of mouse testes,11) it might play a
critical role in the completion of meiosis during male
germ cell differentiation. In contrast, the predicted
protein sequence of GDE5 does not contain a character-
N. YANAKA
istic signal sequence at the N terminus or a trans-
membrane sequence, as determined by hydropathy
analysis, suggesting that GDE5 functions as a cytosolic
protein. Our observations indicate that expression of
GDE5 mRNA is present in mouse skeletal muscle and
cardiac muscle, and is regulated in skeletal muscles of
fasted mice and diabetic KK-Ay mice (unpublished
data). Because the predicted structural feature of GDE5
is an N-terminal carbohydrate binding domain that is
also found in glucoamylases, GDE5 might bind to
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glycogen and might be involved in glucose metabolism
and/or utilization in skeletal muscles. Their diversity of
distribution at the cellular and tissue levels, and
probably enzymatic properties, including substrate
specificity, indicates that GP-PDEs can regulate cellular
functions selectively and can play distinct physiological
roles.
Acknowledgments
I am grateful to all my co-workers, and especially to
Dr. Kenji Omori, for continuous advice and encourage-
ment throughout this work. The initial studies on the
isolation and characterization of GDE3 described here
were performed in the Discovery Research Laboratories
at Tanabe Seiyaku Co., Ltd. I am deeply thankful to Dr.
Norihisa Kato, Dr. Yuji Imai, and Dr. Eri Kawai for
technical advice and helpful discussion, and also thank
the staff, including the graduate and current students
(Yosuke, Zaki and Tome) of this laboratory (Department
of Molecular and Applied Bioscience, Graduate School
of Biosphere Science, Hiroshima University).
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