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Ronald L. Schnaar and Solomon H. SnyderResnick, Phillip B. Storm, Wouter Laroy,John L. Moriarity, K. Joseph Hurt, Adam C. LOCALIZATIONCLONING, CHARACTERIZATION, ANDEnzyme in Proteoglycan Synthesis:UDP-glucuronate Decarboxylase, a KeyTRANSDUCTION:MECHANISMS OF SIGNAL

2002, 277:16968-16975.J. Biol. Chem.

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UDP-glucuronate Decarboxylase, a Key Enzyme in ProteoglycanSynthesis

CLONING, CHARACTERIZATION, AND LOCALIZATION*

Received for publication, September 26, 2001, and in revised form, February 26, 2002Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M109316200

John L. Moriarity‡, K. Joseph Hurt§, Adam C. Resnick§, Phillip B. Storm‡, Wouter Laroy¶,

Ronald L. Schnaar§¶, and Solomon H. Snyder§¶**

From the ‡ Departments of Neurological Surgery, § Neuroscience, ¶ Pharmacology and Molecular Sciences, and Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

UDP-glucuronate decarboxylase (UGD) catalyzes theformation of UDP-xylose from UDP-glucuronate. UDP-xylose is then used to initiate glycosaminoglycan bio-synthesis on the core protein of proteoglycans. In ayeast two-hybrid screen with the protein kinase Akt(protein kinase B), we detected interactions with a novelsequence, which we cloned and expressed. The ex-

pressed protein displayed UGD activity but did notdisplay the activities of homologous nucleotide sugarepimerases or dehydratases. We did not detect phospho-rylation of UGD by Akt nor did we detect any influenceof Akt on UGD activity. Effects of UGD on Akt kinaseactivity were also absent. Northern blot and Westernblot analyses revealed the presence of UGD in multipletissues and brain regions. Subcellular studies and histo-chemistry localized UGD protein to the perinuclearGolgi where xylosylation of proteoglycan core proteinsis known to occur.

Once thought to function only as structural proteins, proteo-

glycans are now known to be crucially involved in a number of

signaling pathways in animals, especially during development(1, 2). Glycosaminoglycan (GAG)1 moieties of proteoglycans are

polymeric, unbranched polysaccharides that can serve as co-

receptors by binding a variety of secreted growth factors such

as fibroblast growth factor, transforming growth factor- /bone

morphogenetic proteins, and hedgehog (3–12). Recent identifi-

cation of several Caenorhabditis elegans mutants deficient in

GAG biosynthesis and transport has demonstrated a critical

role for GAGs in growth and development (13–15).

Proteoglycans are comprised of a core protein covalently

connected to GAG chains. Multiple different GAG chains exist,

including chondrotin sulfate, dermatan sulfate, and heparan

sulfate/heparin. While the disaccharide repeat units compris-

ing the polymeric chains differ, each of these shows a common

form of covalent attachment to the core protein. Specifically,

they attach to a serine of the core protein by a tetrasaccharide

that serves as a primer for polysaccharide growth. This tet-

rasaccharide comprises a xylose (which is O-linked to serine),

galactose, galactose, and glucuronate (Fig. 1). Participation inthe GAG linkage appears to be the sole established function of

xylose in vertebrate biology. Xylose is formed by the decarbox-

ylation of UDP-glucuronate to UDP-xylose (Fig. 1). The UDP

group is removed by xylosyltransferase, which directly attaches

xylose to serine (16). The enzyme activity responsible for the

formation of UDP-xylose via decarboxylation of UDP-glucur-

onate has been characterized for over 30 years. However, de-

spite its importance as a regulatory and possibly rate-limiting

enzyme in GAG biosynthesis, UDP-glucuronate decarboxylase

(UGD) has been difficult to purify and clone, presumably be-

cause of its very low abundance in most tissues. Very recently

a fungal form of the enzyme has been cloned (17). Utilizing a

yeast two-hybrid screen to isolate Akt (protein kinase B) pro-

tein-binding partners/substrates, we identified UGD as an Akt-

interacting protein. In the present study, we report the molec-

ular cloning and characterization of UGD in mammalian

tissues and identify a selective localization to the Golgi.

MATERIALS AND METHODS

Yeast Two-hybrid Screen—Full-length rat Akt was cloned by reversetranscriptase-PCR from adult rat brain total RNA. Utilizing PCR-basedmutagenesis we constructed a triple mutant activated, kinase-dead Akt(Akt*

KD, T308D, S473D, and K179A) (18, 19). Wild type Akt, activated

Akt (Akt*, T308D, S473D), kinase-dead Akt (AktKD

, K179A), and activated kinase-dead Akt (Akt*

KD) were evaluated for expression and

enzymatic activity by GST- or HA-tagged fusion protein plasmid ex-pression in HEK293T cells. The activity characterization agreed withpreviously published data (18, 19). Akt*

KD was subcloned as bait into

yeast expression vector pPC97 containing the GAL4 binding domain

and used to screen a rat hippocampal and cortical cDNA library inpPC86 expressing the GAL4 transactivation domain (20). The plasmidswere introduced sequentially by LiAc-mediated transformation (21)into the PJ69 yeast strain. A total of 1.0 107 independent clones werescreened. Positive interactions were identified by selecting for histidineand adenine prototrophy. Positive clones were further evaluated for-galactosidase expression on plates containing 5-bromo-4-chloro-3-in-

dolyl--D-galactopyranoside (X-gal). Plasmids rescued from positiveclones were retransformed into the HF7C yeast strain (CLONTECH)and once again selected for histidine prototrophy and evaluated for-galactosidase expression by nitrocellulose filter lift assays (21).

Rat Brain cDNA Library Screen and Probe Generation— A rat braincDNA library in ZAPII (Stratagene) was screened per the manufac-turer’s protocol. The probe was generated from the UGD yeast two-hybrid fragment by random priming (Invitrogen) in the presence of [-32P]dATP and dCTP (PerkinElmer Life Sciences). Full-length UGD

* This work was supported by United States Public Health ServiceGrant MH18501 (to S. H. S.), Research Scientist Award DA-00074 (toS. H. S.), and Training Programs in Neuroscience MH20062 (to K. J. H.and A. C. R.) and NS37096 (to R. L. S.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF482705.

** To whom correspondence should be addressed: Dept. of Neuro-science, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore,MD 21205. Tel.: 410-955-3024; Fax: 410-955-3623; E-mail: ssnyder@ bs.jhmi.edu.1 The abbreviations used are: GAG, glycosaminoglycan; DsRed, Dis-

cosoma sp. Red; EYFP, enhanced yellow fluorescent protein; GST, glu-tathione S-transferase; HA, hemagglutinin; HPLC, high performanceliquid chromatography; TDP, thymidine 5-diphosphate; UDP, uridine5-diphosphate; UGD, UDP-glucuronate decarboxylase; ER, endoplas-mic reticulum.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 19, Issue of May 10, pp. 16968–16975, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org16968



was subsequently subcloned into pCMV-HA and pCMV-GST vectors. Epimerase Activity Assay —UDP-galactose-4-epimerase activity was

assayed essentially as described previously (22). Modifications includedthe use of 100 g of lysates from GST-UGD transformed bacteria and0.011 units of UDP-glucose dehydrogenase (Sigma) per a final reaction

volume of 500 l.

Dehydratase Activity Assay —The assay described by Vara et al. (23)was modified by the use of unlabeled TDP-glucose as substrate andNaB3H4 for the reduction of a 4-keto reaction product. Reaction prod-ucts were separated by thin layer chromatography on aluminum-

backed silica plates with a solvent mixture of pyridine, ethyl acetate,and water 26:66:8 (v/v/v). Dried plates were visualized by autoradiog-raphy and stained by spraying with a solution of 10% H

2SO

4 in ethanol

followed by heating for 20 min at 120 °C. Alternatively, the plates werescraped and assayed by scintillation counting.

We also assayed for a dehydratase product spectrophotometrically asdescribed previously (24). Bacterially expressed RmlB (24) was used as

a positive control for dehydratase activity.UDP-glucuronate Decarboxylase Assay —Decarboxylase activity was

assayed as described previously (25). Briefly, lysates from GST-UGD-transformed bacteria or alternatively from transfected HEK293T cells,2 mM NAD, and 750 M unlabeled UDP-glucuronate (Sigma) were

incubated in the presence of 0.2 Ci of uniformly labeled [ glucuronyl-U-14C]UDP-glucuronate (ICN Biomedicals, Inc.). Where indicated, celllysates from untransfected HEK293T were used alone or in combina-

tion with lysates from transfected cells for activity assays. Reactionbuffer consisted of 0.1 M NaH

2PO

4, 0.5 g/liter EDTA, and 5 ml/liter

-mercaptoethanol. Detection of UDP-glucuronate decarboxylase activ-ity was based on the detection of released 14CO2. During the reaction,released 14CO

2 was trapped in an inner tube containing 0.2 ml of

ethanolamine and 2-methoxyethanol, 1:2 (v/v). Released 14CO2 wasthen determined in a liquid scintillation counter.

HPLC —Enzyme reactions for HPLC analysis were conducted as

described above utilizing 25,000 –50,000 cpm of uniformly labeled [ glu-

curonyl-U-14C]UDP-glucuronate. Uniformly labeled [ xylose-U-14C]UDP-xylose was obtained from American Radiolabeled Chemicals Inc.

Assays were quenched with ice-cold perchloric acid and neutralized asdescribed previously (26). Where indicated, the enzyme was heat-inac-tivated by incubation for 5 min at 100 °C. HPLC analysis of reaction

products and standard was conducted using a Zorbax SAX column(Hewlett-Packard) that was eluted with a gradient generated by mixing solution A (1 mM Na

2EDTA) and buffer B (solution A plus 1.3 M

(NH4)2HPO4, pH 3.85 with H3PO4) as follows: 0 –5 min, 0% buffer B;5– 65 min, 0 –30% buffer B; 65–75 min, 100% buffer B; 75–90 min, 0%

buffer B. The fractions (0.5 ml) were collected and counted using 5 ml of Ultima-Flo AP LCS mixture (Packard, Downers Grove, IL).

Electrospray Mass Spectrometry —Enzyme reactions were conductedas described above with 1 mg of lysates from GST-UGD-transformed

bacteria. Alternatively, the enzyme was heat-inactivated by incubationfor 5 min at 100 °C. Samples were desalted and prepared for massspectrometry analysis of nucleotide sugars by charcoal adsorption and

elution as has been described previously (27). Samples were diluted inthe elution solvent (ethanol/water/concentrated ammonia, 600/400/6.5(v/v/v)) so that a 30 M concentration of original UDP-glucuronate (as

calculated from starting material) was obtained. This was then directlyinjected into a PE SCIEX API 150EX mass spectrometer with an elec-trospray source at a flow rate of 9 l/min. Negative ion spectra were

obtained.

FIG. 1. Schematic representation of UDP-xylose synthesisfrom UDP-glucuronic acid by UGD (*). Enzymatic attachment of UDP-xylose to the core protein (via a serine hydroxyl) then initiatesproteoglycan synthesis by the formation of the linker tetrasaccharideand subsequent GAG attachment and elongation.

FIG. 2. Full-length nucleotide and amino acid sequence for rat UGD. Underlined sequences represent hydroxyl-containing amino acids

and an Y XXX K motif potentially involved in substrate and NAD

binding, respectively (see text for details).

Cloning of UDP-glucuronate Decarboxylase 16969



Northern Analysis — A multiple tissue Northern blot membrane(CLONTECH) containing 2 g of mRNA/lane was hybridized using Expresshyb (CLONTECH) according to manufacturer’s instructionswith a probe generated from the yeast two-hybrid fragment by randompriming (Invitrogen) in the presence of [-32P]dATP and dCTP(PerkinElmer Life Sciences). Multiple cell line blots were prepared fromcell lysates using Trizol (Invitrogen) as per the manufacturer ’s protocol.Each lane contained 20 g of total RNA. Equal amounts of ribosomal

RNA and -actin were present in each lane.Transient Transfections and Bacterial Transformations for UGD Ac-

tivity Assay —HEK293T cells were transiently transfected using Lipo-

fectAMINE 2000 (Invitrogen) as per the manufacturer’s protocol. Cellswere lysed (after 48 h) in the following buffer: 50 mM Tris-HCl, pH 7.5,0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM

-glycerol phosphate, 5 mM pyrophosphate, 1 mM NaOV 4

, 0.1% -mer-captoethanol, 1 M microcystin. Lysates were briefly sonicated, centri-fuged at 16,000 g for 15 min, and the supernatant used in activityassays.

DH5 subcloning efficiency Escherichia coli (Invitrogen) were trans-

formed with a GST-UGD plasmid, and fusion protein expression wasinduced as per the manufacturer’s protocol. Cell lysates were sonicated,

centrifuged at 16,000 g for 15 min, and the supernatant used in

FIG. 3. CLUSTAL-W alignment of UGD (top) and putative orthologs in M. musculus (GenBankTM accession number AAK85410),C. elegans (GenBankTM accession number T15892), D. melanogaster (GenBankTM accession number AAF50474), F. neoformans(GenBankTM accession number AAK59981), and P. sativum (GenBankTM accession number BAB40967).

Cloning of UDP-glucuronate Decarboxylase16970



activity assays or snap-frozen and kept at 80 °C for up to 3 monthswithout loss of activity.

In Vitro Binding Assays —HEK293T cells were co-transfected using

LipofectAMINE 2000 (Invitrogen) with GST-UGD and HA-AKT, HA- AKT

KD, HA-AKT*, or an unrelated fusion protein designated HA-X.

Similar experiments were performed with HA-UGD and the corre-

sponding GST-Akt plasmids (data not shown). After 48 h, cells werelysed in the followingbuffer: 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM

EDTA, 2 mM EGTA, 0.1% Triton X-100, 0.1% Nonidet P-40, 50 NaF, 20mM -glycerol phosphate, 1 mM dithiothreitol, 100 mg/liter phenylmeth-ylsulfonyl fluoride, and protease inhibitor mixture (Sigma). Lysateswere briefly sonicated and centrifuged at 16,000 g for 15 min. Aftercentrifugation, 1.0 mg of the supernatants was incubated with a 50%slurry of prepared glutathione-Sepharose (Amersham Biosciences) for1.5 h at 4.0 °C with slow rotation. Samples were washed five times withphosphate-buffered saline (Invitrogen). The agarose was resuspendedin sample loading buffer, separated by SDS-PAGE, and immunoblotted

using the indicated antibodies. Anti-GST (Sigma) and anti-HA (Co- vance) primary antibodies were both used at dilutions of 1:2000.

Antibody Generation — A rabbit polyclonal antibody was raised

against GST-tagged UGD yeast two-hybrid fragment (Covance). Theantibody was affinity-purified as described previously (28). Immuno-blots were performed with a primary antibody dilution of 1:1000.

Subcellular Fractionation —Brains from five male adult rats werehomogenized in 100 ml of 0.32 M sucrose with a glass/Teflon homoge-nizer. Homogenate was centrifuged for 10 min at 800 g to give pellet(P1) and supernatant (S1). S1 was centrifuged for 15 min at 9200 g togive pellet (P2) and supernatant (S2). S2 was centrifuged for 90 min at

100,000 g to give pellet (P3) and supernatant (S3). The P2 fractionwas resuspended in 3 ml of 0.32 M sucrose and hypotonically lysed in 27ml of ice-cold water. Lysate was homogenized with a glass/Teflon ho-mogenizer. Hepes (2 M; pH 7.4) was added to a final concentration of 50mM, and the sample was centrifuged for 20 min at 25,000 g to givepellet (LP1) and supernatant (LS1). LS1 was centrifuged for 90 min at165,000 g to give pellet (LP2) and supernatant (LS2). Protein con-centration of the fractions was determined, and 12 g of protein fromeach fraction was separated by SDS-PAGE followed by immunoblot.

In Vivo Enzyme Localization —HEK293T cells were transiently co-transfected with a total of 2 g of pDsRed-UGD and either pEYFP-Golgi

or pEYFP-ER (CLONTECH) using LipofectAMINE 2000 according tothe manufacturer’s directions (Invitrogen). pEYFP-Golgi encodes a fu-sion protein consisting of enhanced yellow fluorescent protein (EYFP)and a sequence encoding the NH2-terminal 81 amino acids of human-1,4-galactosyltransferase; pEYFP-ER encodes a fusion protein con-sisting of EYFP, the ER targeting sequence of calreticulin, and the ERretrieval sequence KDEL. Cells were grown on four-well glass micro-scope slides and were processed for enzyme localization studies 48 hafter transfection. Photomicrographs shown in Fig. 10 were obtainedwith a Nikon Eclipse TE300 inverted microscope and captured withOpenlab (Improvision).

RESULTS

Molecular Cloning of UGD, an Akt Yeast Two-hybrid Inter-

actor —In an effort to identify protein-binding partners of the

signaling protein kinase Akt (protein kinase B), we utilized a

yeast two-hybrid analysis with an activated, but kinase-dead,

form of Akt as bait. We identified two interactors, one of which

FIG. 4. Augmented evolution of 14CO2

from [ glucuronyl-U-14C]-labeled UDP-glucuronate with increasing amounts of UGD.Each of the four reactions utilized a total of 1 mg of cell lysate with

varying proportions of transfected (GST-UGD) and untransfected(293T) lysates. Evolved 14CO2 was determined in a liquid scintillationcounter. These results were reproduced in nine similar experimentswith both mammalian and bacterial cell lysates.

FIG. 5. HPLC analysis of UGD reaction products. A, enzymereaction with native UGD utilizing uniformly labeled [ glucuronyl-U-14C]UDP-glucuronate. B, HPLC elution of uniformly labeled [ xylose-U-14C]UDP-xylose standard. C, heat-inactivated enzyme reaction utiliz-ing uniformly labeled [ glucuronyl-U-14C]UDP-glucuronate. HPLCelution fractions (0.5 ml) were collected and counted in a liquid scintil-lation counter. These results were reproduced in six separateexperiments.




remains uncharacterized. The other interactor, which we de-

termined to be UGD, was identified six times in the two-hybrid

screen. A rat brain cDNA library was screened utilizing the

UGD yeast two-hybrid fragment as probe. Four interacting

library fragments were recovered. A full-length cDNA sequence

of UGD was assembled from two of the rat brain cDNA frag-

ments, the yeast two-hybrid fragment (encompassing amino

acids 32– 420), and database analysis (Fig. 2). The starting

methionine was assigned to the first in-frame AUG codon pre-

ceded by an upstream stop codon. The full-length open reading

frame of UGD contains 1260 nucleotides coding for a protein of

420 amino acids with a predicted molecular mass of 47 kDa

(Fig. 2). Data base analysis revealed varying homology with

putative and established UDP-glucuronate decarboxylases, nu-

cleotide sugar epimerases, and nucleotide sugar dehydratases.

Based on homology with UDP-galactose-4-epimerase, regions

of UGD were identified that may be important in substrate and

NAD binding. Specifically, UDP-galactose-4-epimerase and

UGD contain a critical threonine or serine required for binding

to the 4-hydroxyl group of the sugar substrates. Also, a con-

served Y XXX K motif for binding to NAD is present in both

UGD and UDP-galactose-4-epimerase (29) (Fig. 2). Data base

analysis suggested that UGD is highly conserved across a wide

evolutionary range from plants to mammals (Fig. 3). Hydrop-

athy plot analysis (30) of the UGD protein indicated a single

putative transmembrane domain at residues 20 –38. Consist-

ent with a type II transmembrane protein topololgy, the anal-

ysis predicted a short NH2-terminal segment most likely pro-

truding into cytoplasm and a longer luminal carboxyl-terminal

domain. Such a topology also exists in the putative homologs of

UGD in Mus musculus, C. elegans, and Drosophila melano-

gaster but not in Filobasidiella neoformans or Pisum sativum.

Identification of UDP-glucuronate Decarboxylase Activity —

Based on sequence similarities, we assayed our cloned, ex-

pressed protein for UDP-galactose-4-epimerase activity, dTDP-

glucose-4,6-dehydratase activity, and UGD activity. We

assayed for epimerase activity by spectrophotometrically mon-

itoring the production of UDP-glucose from UDP-galactose viacoupling to NAD in the presence of UDP-glucose dehydrogen-

ase (22). Our clone failed to demonstrate any epimerase activ-

ity in this assay (data not shown).

We examined dehydratase activity (conversion of dTDP-glu-

cose to dTDP-4-keto-6-deoxyglucose) in two ways. First, we

reduced the 4-keto product of the reaction with tritiated NaBH4

and separated the reaction mixture with silica TLC. With di-

rect visualization, autoradiography, and scintillation counting,

we were unable to detect production of any tritiated, 6-deoxy

reaction products by our clone. We also spectrophotometrically

assayed for the production of the 4-keto product (24, 31), but

were unable to detect any product in the presence of our clone

(data not shown).

We monitored UGD activity in three ways. Employing uni-formly labeled [ glucuronyl-U-14C]UDP-glucuronate, we meas-

ured the evolution of 14CO2

(25). In this assay we detected

robust, dose-dependent activity of our GST-tagged clone in

transiently transfected HEK293T cell lysates (Fig. 4). Gluta-

thione precipitation and thorough washing confirmed that the

activity resulted from our clone (data not shown). This activity

obeys Michaelis-Menten kinetics with a K m

near 1 mM (data not

shown). As the starting material was uniformly labeled on the

glucuronyl moiety, we could not assert definitively that the

evolved CO2 originated from the decarboxylation of the 6-car-

bon. To further characterize the biosynthesis of UDP-xylose by

our clone, we incubated GST-tagged clone with uniformly la-

beled [ glucuronyl-U-14C] UDP-glucuronate and separated the

reaction products by HPLC. In the presence of boiled GST-

tagged enzyme (Fig. 5C) or an unrelated GST-tagged protein

(data not shown), only a single sharp peak appeared with an

elution time corresponding to that of the starting material.

However, incubation with our native, tagged clone resulted in

the appearance of a second sharp peak (Fig. 5 A) with an elution

time identical to that of authentic uniformly labeled [ xylose-U-14C]UDP-xylose (Fig. 5 B). To obtain definitive evidence that

UDP-xylose was the product of our assay, we performed elec-

trospray mass spectrometry. Negative ion spectra of reaction

mixtures in the presence of heat-inactivated enzyme (Fig. 6 B)

only showed peaks corresponding to UDP-glucuronate ([M-H]

579.3), NAD ([M-H] 662.3), and a NAD degradationproduct ([M-H] 540; loss of niacinamide). In the presence of

our putative UGD, an additional peak appeared corresponding

to the mass of UDP-xylose ([M-H] 535.2) (Fig. 6 A). Minor

peaks were evident corresponding to the masses of mono- and

disodiated UDP-glucuronate and UDP-xylose.

Molecular and Functional Analysis of Akt/UGD Interac-

tions —Since UGD was cloned based on its interactions with

Akt, we wondered whether such interactions are physiologic. In

HEK293T cells transfected with GST-UGD and HA-Akt or with

HA-UGD and GST-Akt, pull-down of GST-Akt brought down

HA-UGD (data not shown) and pull-down of GST-UGD brought

down HA-Akt, while an unrelated protein did not co-precipitate

with UGD (Fig. 7).

We wondered whether there are any functional interactions

FIG. 6. Electrospray negative ion spectra of enzyme reactionproducts with active UGD ( A) or with heat-inactivated UGD ( B).In addition to the UDP-glucuronate peak ([M-H] 579.3), a secondsignificant peak appeared in the analyses reflecting the presence of monosodiated UDP-glucuronate ([MNa-2H] 601.3). Reactionswith heat-inactivated enzyme ( B) also displayed mass peaks corre-sponding to NAD ([M-H] 662.3) and its degradation product ([M-H] 540.4) (reflecting the loss of niacinamide from NAD). Analysis

of reactions containing active UGD ( A) revealed additional peaks cor-responding to UDP-xylose ([M-H] 535.2) and monosodiated UDP-xylose ([MNa-2H] 557.1). y axis scale is in millions.




between Akt and UGD. We have been unable to demonstrate

alterations in Akt activity with UGD transfection in basal,

serum-starved, or insulin-like growth factor-stimulated

HEK293T cells (data not shown). Although UGD does not con-

tain the canonical Akt phosphorylation motif (R X R XX S/T X )

present in many known Akt substrates (32), we examined the

possibility of Akt phosphorylation of UGD. We were unable to

detect phosphorylation of GST-UGD by activated HA-Akt in an

in vitro kinase reaction nor did we detect any influence of

activated HA-Akt on UGD activity (data not shown).

Tissue Localization of UGD —Northern analysis revealed the

highest densities of UGD mRNA in heart, brain, and testes

(Fig. 8 A). Substantial levels were also evident in kidney, liver,

and lung with much lower densities in spleen and skeletal

muscle. Northern analysis of transformed cell lines revealed

substantial levels of UGD mRNA in 3T3, RBE7, and PC12 cell

lines but little to no transcript in 293T, HeLa, COS-1, Jurkat,

or 9L glioma cell lines (Fig. 8 B). In all of these tissues and cell

lines we detected a single transcript of about 2.1 kb, suggesting

that there is no significant alternative splicing.

For immunochemical studies we developed a rabbit poly-

clonal antibody to GST-UGD. The antibody recognized a bandof 47 kDa in transiently transfected cells (data not shown) as

well as in native rat tissues (Fig. 9). Additionally, in all tissues

an additional band of about 70 kDa was evident (data not

shown). Kidney samples contained a second 38-kDa cross-reac-

tive band recognized by the antibody. Preabsorption of purified

antibody with GST-UGD eliminated the 47-kDa band in all

tissues. Preabsorption of purified antibody with GST alone or

agarose beads did not alter the 47-kDa band (Fig. 9 A).

UGD protein was most enriched in kidney, liver, and brain

with negligible staining in Western blots of heart, spleen, skel-

etal muscle, lung, and testes (Fig. 9 B). This pattern differed

from the distribution of UGD mRNA, which was highly concen-

trated in testes and heart (Fig. 8 A). Within the central nervous

system, UGD protein distribution varied with the highest

amounts in the cerebellum, thalamus, and spinal cord and

much lower levels in cerebral cortex, hippocampus, corpus stri-atum, and olfactory bulb (Fig. 9 B).

Subcellular Localization of UGD —The addition of xylose in

the biosynthesis of GAGs begins in the late endoplasmic retic-

ulum and/or at the ER-to-Golgi interface and continues in the

Golgi (33–35). Furthermore, UGD activity has been found in

chick chondrocytes to co-localize with xylosyltransferase activ-

ity in subcellular fractions (35). To evaluate the intracellular

localization of UGD, we conducted subcellular fractionations

(Fig. 9C). UGD protein was predominantly particulate with

highest densities in the crude microsomal fraction of whole

brain (P3). To assess localization to synaptic vesicle fractions

and other membranes within nerve terminals, we lysed the

synaptosome-nerve terminal containing P2 fraction, which pro-

vides the LP1 (lysate) and LS1 (lysate supernatant fractions).

FIG. 7. Western blot analysis of GST-UGD and HA-Akt co-pre-cipitation experiments. Glutathione bead pull-down of GST-taggedUGD co-precipitated HA-AKT from 1 mg of cell lysates from HEK293

cells transiently co-transfected with GST-UGD and HA-AktWT, HA- Akt

KD, or HA-Akt*

WT. Glutathione pull-down of GST-tagged UGD from

transiently co-transfected cells failed to co-precipitate an unrelated,HA-tagged protein ( HA-X ). Load: HA-tagged protein content in 20 g of transfected cell lysates; Pulldown: GST-UGD bound to glutathione-Sepharose beads after incubation with transfected cell lysates; Co-

precipitate: HA-tagged proteins co-precipitating with glutathione-Sepharose-bound GST-UGD.

FIG. 8. Expression pattern of UGD in multiple rat tissues ( A)and multiple cell lines ( B) determined by Northern blot analy-sis. A single 2.1-kb transcript is identified in multiple tissues and celllines.




Centrifugation of LS1 yielded a supernatant LS2 fraction and a

crude synaptic vesicle fraction, LP2. UGD was detected in LP2

and LS2 but not in LP1 fractions.

To assess the intracellular localization of UGD in intact cells,

we conducted histochemistry. Because of the low levels of en-

dogenous UGD, we were unable to conduct immunohistochem-

istry for native UGD. Accordingly, we transfected HEK293

cells with DsRed2-labeled UGD and compared its localization

with co-transfected EYFP-ER and EYFP-Golgi, fusion protein

markers for the endoplasmic reticulum and Golgi, respectively.

We observed intense staining for UGD in the perinuclear Golgi

(Fig. 10).

DISCUSSION

In the present study, we present the molecular cloning and

characterization of mammalian UGD. UGD is evolutionarily

highly conserved with 75– 80% amino acid sequence identity

and 90% similarity between plants and mammals. Although we

discovered UGD based on its binding to Akt, we have failed to

thus far identify any major regulatory interactions between the

two proteins.

Data base analysis revealed similarity of UGD to UDP-ga-

lactose-4-epimerase and TDP-glucose-4,6-dehydratase. These

similarities may be based on properties shared by UGD and

these other enzymes. These enzymes all employ NAD as a

co-factor and bind UDP or TDP sugars. All of them are hypoth-

esized to share an initial catalytic step involving oxidation of

the 4-carbon of the sugar to a ketone. This is the sole function

of the sugar dehydratase. The epimerase asymmetrically re-

duces the 4-ketone to stereospecifically produce the C-4 epimer,

while UGD is thought to proceed through -decarboxylation of

the 6-carbon followed by sterospecific reduction of the 4-carbon

(28, 29).

Subcellular fractionation and histochemical studies localized

UGD primarily to the Golgi. Consistent with this, hydropathyplot analysis of the protein sequence indicated a putative type

II transmembrane topology common to Golgi-localized proteins

(36). Previous studies of UGD activity in cultured chick chon-

drocytes as well as mouse mast cell tumors have localized the

activity to the particulate cell fractions (35, 37, 38). Studies of

xylose biosynthesis and xylosylation in intact cells, which mon-

itor transformation of UDP-glucuronate to UDP-xylose in per-

meabilized chick chondrocytes, indicate predominant localiza-

tions to the Golgi and endoplasmic reticulum (34, 35).

Consistent with our hydropathy analysis, these studies evalu-

ated the possible luminal or cytosolic orientation of membrane-

associated enzyme activity by monitoring trypsin sensitivity

and found UGD activity was lost in the presence of trypsin only

when organelle membranes were disrupted (35). While UDP-

FIG. 9. Antibody characterization and UGD distribution in rattissues. Western blot demonstration that preabsorption of rabbit poly-clonal anti-UGD antibody with GST-UGD resulted in the disappear-ance of the 47-kDa band, while preabsorption with GST or agarosebeads alone did not ( A). B , multiple tissue and multiple brain regionWestern blots depicting tissue and central nervous system distribution

of UGD are shown (CTX , cortex; CBL, cerebellum; HC, hippocampus; STR, corpus striatum; OB, olfactory bulb; Thal, thalamus; SC, spinalcord). C, Western blot analysis of subcellular fractions demonstrating predominance of UGD in P3, consistent with Golgi localization.

FIG. 10. Localization of UGD in intact, living cells. HEK-293Tcells were co-transfected with DsRed2-UGD and either EYFP-ER ( A –C)or EYFP-Golgi ( D – F ). EYFP-ER encodes a fusion protein consisting of enhanced yellow fluorescent protein, the sequence encoding the endo-plasmic reticulum targeting sequence of calreticulin, and the sequenceencoding the ER retrieval sequence (KDEL). EYFP-Golgi encodes afusion protein consisting of enhanced yellow fluorescent protein and asequence encoding the N-terminal 81 amino acids of human -1– 4-

galactosyltransferase. EYFP-ER expression ( A) and DsRed2-UGD ( B)displayed some overlap (C). Expression of EYFP-Golgi ( D) and DsRed2-UGD ( E) displayed nearly identical co-localization, suggesting predom-inately perinuclear Golgi localization of UGD. Scale bar shown corre-sponds to 10 m.




xylose transport into the Golgi has been described (33, 35), it

has been suggested to be the result of nonspecific nucleotide

sugar transport associated with other nucleotide sugar trans-

porters (35). In contrast to our cloned sequence and its mam-

malian, C. elegans, and D. melanogaster homologs, hydropathy

plot analysis of homologous sequences of plant and fungal

origin showed no predicted membrane spanning domains; con-

sistent with this, previous studies of plant and fungal UGD

activity have found them to be associated with soluble cell

fractions (39, 40). Indeed, the recent cloning of UGD from the

pathogenic fungus Cryptococcus neoformans found the ex-

pressed enzyme to localize primarily to soluble cell fractions

(17).

The enzymes crucial for GAG biosynthesis and their impor-

tance for proper growth and differentiation have been well

characterized in C. elegans (13–15). In this species, epithelial

invagination of the vulva is a major, developmentally regulated

event. Screening for mutations that interfere with this process

led to the identification of eight specific “squashed vulva”

( SQV ) genes (14). Seven of these eight genes have been cloned.

Notably, all are involved in GAG biosynthesis or transport (13,

15, 41). SQV -1 is the only one of these not yet cloned. C. elegans

genetic map data base analysis reveals a close proximity of a

UGD ortholog to SQV -1 on chromosome IV. It is likely thatUGD and SQV -1 are identical. If so, this would imply a critical

role for UGD in embryonic development.

Acknowledgments —We thank Bahman Aghdasi, Jamie Cheah,Christopher Ferris, Adolfo Saiardi, Kristin Whitford, Keqiang Ye, and

Alec Resnick for technical assistance and insightful comments. We arealso indebted to Michael McNeil for generous contribution of RmlBtransformed bacteria for use as positive controls in our dehydrataseassays.

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