Roles of the TPR domain in O-GlcNAc Transferase …cells (20,21). The OGT gene resides on the X chromosome near the centromere (Xq13 in man), and its targeted deletion results in embryonic

Roles of the TPR domain in O-GlcNAc Transferase (OGT) Targeting and

Protein Substrate Specificity

Sai Prasad N. Iyer 1,2,4 and Gerald W. Hart 1,3

Running title: TPR-mediated substrate specificity and targeting of OGT

1 Department of Biological Chemistry, Johns Hopkins University School of Medicine,

Baltimore, MD 21205-2185, USA

2 Graduate Program, Department of Biochemistry and Molecular Genetics, Univerisity

of Alabama at Birmingham, Birmingham, AL 35294, USA

3 Address Correspondence to:

Gerald W. Hart

Department of Biological Chemistry

Johns Hopkins University School of Medicine

725 North Wolfe Street

Baltimore, MD 21205-2185

TEL: (410) 614-5993; FAX: (410) 614-8804 Email: [email protected]

4 Present Address: Schering-Plough Research Institute

2015 Galloping Hill Road

K-15-3-3600; Room C332c

Kenilworth, NJ 07033-0530

USA

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 30, 2003 as Manuscript M300036200 by guest on February 15, 2020

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Summary

The abundant and dynamic post-translational modification of nuclear and

cytosolic proteins by O-linked N-Acetylglucosamine (O-GlcNAc) is catalyzed by O-

GlcNAc transferase (OGT). Recently, we reported the identification of a novel family of

OGT-interacting proteins (OIP) that interact strongly with the TPR domain of OGT

(Iyer, S., et.al (2003) J. Biol. Chem. 278, 5399-5409). Members of this family are

modified by O-GlcNAc and are excellent substrates of OGT. Here we further

investigated the role of the TPR domain in the O-GlcNAcylation of OIP106, one of the

members of this OIP family. Using N terminal deletions, we first identified the region of

OIP106 that binds OGT, termed the OGT Interacting domain (OID). Deletion analysis

then indicated that TPR repeats 2-6 of OGT interact with the OID of OIP106. Apparent

Km of OGT for the OID of OIP106 equals 3.35 µM. Unlike small peptide substrates, the

glycosylation of OID is dependant upon its interaction with the first 6 TPRs of OGT.

Furthermore, the isolated TPR domain of OGT competitively inhibits the glycosylation of

the OID protein, but does not inhibit the glycosylation of a twelve amino acid CK II

peptide substrate, providing kinetic evidence for the TPR domain�s role as a protein

substrate docking site. Additionally, both the OID of OIP106 and nucleoporin p62

compete for each other for glycosylation by OGT. These studies support the model that

the catalytic subunit of OGT achieves both high specificity and a remarkable diversity of

substrates by complexing with a variety of targeting proteins via its TPR protein-protein

interaction domains.

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Introduction

Post translational modification of proteins by enzymatic addition of single O-

linked N-acetylglucosamine (O-GlcNAc) moieties to the hydroxyl groups of ser/thr

residues been shown to be a major form of protein regulation (2-4). This modification is

known to occur on key proteins that modulate cell function, such as RNA polymerase II

(5, 33), transcription factors (6-8), cytoskeletal (9,10), heat shock (11, 34), oncoproteins

(12, 35) and various kinases and phosphatases (13,14). Attachment of O-GlcNAc (O-

GlcNAcylation) is a dynamic (added/removed in minutes) and ubiquitous modification

and occurs in all higher eukaryotes, including plants and animals. Since many of the sites

that are modified by O-GlcNAc are either the same or are adjacent to sites of ser/thr

phosphorylation (15), O-GlcNAcylation of proteins is thought to play a regulatory role,

analagous to protein regulation by phosphorylation (3).

Enzymes that catalyze addition and removal of O-GlcNAc on and off proteins are

analagous to those that catalyze phosphorylation i.e kinases and phosphatases.

O-GlcNAc transferase (OGT) (EC 2.4.1, uridine diphospho-N-acetylglucosamine:poly-

peptide β-N-acetylglucosaminyltransferase) is an enzyme that transfers the GlcNAc to

proteins from a UDP-GlcNAc sugar donor. Conversely, an O-GlcNAc specific β-N-

acetylglucosaminidase (O-GlcNAcase) removes the sugar from O-GlcNAcylated

proteins. Both enzymes have been purified, characterized and cloned (16-21). Both

enzymes are highly conserved throughout evolution, with species orthologs present from

Caenorhabditis elegans (C.elegans) through man (18,20,21).

The 110 kDa OGT enzyme is a highly unique and ubiquitous glycosyltransferase

which is encoded by a single gene and is present in both nucleus and the cytoplasm of


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cells (20,21). The OGT gene resides on the X chromosome near the centromere (Xq13 in

man), and its targeted deletion results in embryonic lethality in mice (22), indicating that

it is essential for life. Recently, the previously reported partial human OGT clone

described by Lubas et al., (21) has been characterized as an alternatively spliced isoform

of OGT that targets specifically to the mitochondria (mOGT) (40). Thus two distinct

isoforms of OGT exist; a nucleocytoplasmic form of OGT and its alternatively spliced

mitochondrial isoform. Interestingly, the mOGT isoform is not active in the

mitochondria toward mitochondrial protein substrates, suggesting that it may have

another function (40). Sequence analysis of the 110 kDa enzyme showed it to contain

two distinct modular halves. In its amino terminal half, OGT contains a TPR protein-

protein interaction domain, whereas the unique carboxy half of the enzyme is known to

contain its catalytic domain (20,21). The nucleocytoplasmic OGT enzyme contains

eleven and a half tetratricopeptide repeats (TPR), whereas the mOGT isoform contains 9

TPRs, preceded by a mitochondrial targeting sequence (20, 21, 40) and these domains

have been shown to mediate protein-protein interactions in a variety of proteins (23, 36,

37).

Various functions have been attributed to the TPR domain of OGT. The native

OGT holoenzyme exists as a trimer and the TPR domain has been shown to be required

for its trimerization (24). The TPR domain has also been shown to mediate substrate

specificity in a variety of peptide substrates (24). Furthermore, recently it was shown that

the TPR domain is also responsible for targeting OGT to mSin3A transcriptional

repressor complexes (25). While much is known about OGT�s peptide substrate


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specificity, it is unclear as to how the substrate specificity of its physiological protein

substrates is regulated by the TPR domain.

We have recently identified a novel family of coiled-coil domain proteins that

interact with OGT (1). GRIF-1 and OIP106, the two archetypical members of this

family, interact with the TPR domain strongly. GRIF-1 is a GABAA receptor associated

protein (26) and is thought to play a role in targeting OGT to GABAA receptor complexes

to mediate GABA signaling cascades. OIP106 was shown to associate in a complex with

RNA polymerase II, suggesting a role in targeting OGT to RNA Pol II complexes for

transcriptional machinery O-GlcNAcylation (1). Interestingly, both proteins are modified

by O-GlcNAc and are substrates for OGT (1).

Here, we investigated the O-GlcNAcylation of OIP106, to gain further insights

into the enzymatic mechanisms regulating the specificity of OGT. Specifically, we

wanted to study how the TPR domain functioned in relation to substrate specificity of

OGT to a bonafide physiological protein substrate such as OIP106. We localized the

domains of OGT and OIP106 interactions and found that the OGT interaction domain

(OID) of OIP106 is a high affinity substrate for OGT. Furthermore, deletion of specific

TPRs of OGT results in a loss of binding and consequently, loss of O-GlcNAcylation of

OID. Free TPR domain effectively inhibits the glycosylation of OID, but not the

glycosylation of small peptides, in a dose-dependant manner, providing kinetic evidence

that the TPRs function as protein substrate �docking� sites. In addition, both

recombinant nucleoporin p62 (a high affinity substrate) and OID proteins effectively

competed with each other for glycosylation by OGT. We propose a model for the

possible mechanism of OGT glycosylation toward its various protein substrates.


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Experimental Procedures

Plasmid constructs: The baculovirus expression constructs encoding for rat OGT and its

TPR truncations have been described previously (24). Constructs encoding for an N-

terminal hexa-His tagged TPR (pRSET-TPR) were generated by subcloning the TPR

fragments into the pRSET vector (Invitrogen). The pCITE-OIP106 construct used for in

vitro transcription/translation reactions has been previously described (1). N-terminal

OIP106 deletion constructs were generated by PCR of the appropriate regions and

subcloning into pCITE4c. The OID of OIP106 was generated by PCR of the region of

OIP106 encoding for residues 639 through 890 and cloned into pET32 for overexpression

in E.coli as an S/His-tagged fusion protein. The GST-p62 construct was generated by

subcloning the p62 insert into pGEX 5x-1 (Pharmacia).

Protein Expression and Purification: Unless indicated otherwise, the E.coli strain BL21

(DE3) Codon plus RIL strain (Stratagene) was used for the overexpression of TPR and

OIP106 protein constructs in either LB or DYT-amp. Unless indicated otherwise, all

purified proteins were desalted in 20 mM Tris pH 7.8, 20-40% (v/v) glycerol (v/v),

0.02% (w/v) sodium azide and stored in -20°C. His-tagged TPR protein (pRSET-TPR)

was overexpressed and purified under native, non-denaturing conditions via nickel

affinity chromatography. Baculovirus produced recombinant full length OGT, ∆2.5 and

∆5.5 TPR deletion constructs were overexpressed and purified as described before (24).

S-tagged OID was overexpressed and purified via S-protein affinity

chromatography under mildly denaturing conditions in the presence of 1.3 M urea.

Insoluble S-OID protein was extracted by incubating the insoluble cell pellet in 6 M

urea/TBST (20 mM Tris pH 8, 150 mM NaCl, 0.1% (v/v) Triton X-100) for 1 h at 4°C.


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The extract was spun at 39 000 xg for 20 min at 4°C and the supernatant containing S-

OID was diluted down to 1.3 M urea by the addition of TBST. The resulting 1.3 M urea

supernatant was loaded onto S-protein agarose (Novagen) equilibrated in 1.3 M

urea/TBST and S-OID was bound by incubation for 30 min, washed extensively and used

for subsequent assays. Alternatively, the 1.3 M urea/TBST supernatant was passed over

Talon IMAC resin (Clontech) and S-OID was purified according to manufacturer�s

instructions.

GST-p62 was expressed in the E.coli strain BL21 (DE3) RP (Stratagene).

Overexpressed GST-p62 was purified essentially as previously described (38). Purified

recombinant GST-p62 was found to be 90-95% pure as judged by SDS-PAGE analysis

(data not shown).

Antibodies and western blot analysis: Anti-OGT antibodies AL25 (20) and AL28 (1)

were used at a final concentration of 25-50 ng/ml in 5% (w/v) milk in TBS containing

0.05% (v/v) Tween-20 for 16 h at 4°C. S-protein HRP conjugate (Novagen) was used at

1/5 000 according to manufacturer�s instructions. Anti-O-GlcNAc antibody monoclonal

CTD 110.6 was used at 1/2 500, as described previously (39). All blots were developed

with the enhanced chemiluminescence (ECL) reagent (Pharmacia).

Protein-Protein Interaction Assays: Wildtype OIP106 and and its N terminal truncated

mutants (for OID localization studies) were synthesized with either 35S-Met or unlabeled

amino acids in TnT in vitro transcription and translation rabbit reticulocyte lysates

(Promega) with an S-tag fusion, which is encoded by their respective pCITE vectors.

Following synthesis, the reactions were diluted in HBS containing 0.3 M NaCl and S-

tagged OIP106 proteins were bound to S-protein agarose for 3 h at 4°C. The beads were


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washed extensively in their respective binding buffer and eluted by boiling in SDS-PAGE

sample buffer. Samples were then analyzed by western blot with either S-protein HRP

and AL28. The OID-OGT interaction assays were performed as follows. Recombinant

baculovirus produced full length OGT, ∆2.5 OGT, ∆5.5 OGT and E.coli His-TPR

proteins were incubated with S-OID in Tris buffered saline (TBS) containing 20 mM Tris

pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% (w/v) Triton X-100 for 3.5 h at

4°C. Proteins were then pulled down with S-protein agarose, washed extensively in

binding buffer and eluted by boiling in SDS-PAGE sample buffer. Samples were then

analyzed by western blot with S-protein HRP and AL25.

OGT Activity Assays: OGT activity assays were performed essentially as described (13)

with minor modifications. The S-OID protein used as a substrate for OGT, was either in

a soluble form (IMAC purified) or bound to S-protein agarose. OGT protein glyco-

sylation assays were performed by incubating recombinant baculovirus produced full

length OGT, ∆2.5 OGT or ∆5.5 OGT as enzyme sources. 1 µg of the indicated OGT

protein was incubated with the indicated amounts of S-OID protein substrate, 1 to 1.5 µCi

UDP-[3H]GlcNAc (34 Ci/mmol, NEN) in 50 mM Tris pH 7.4, 12.5 mM MgCl2, 1 mM

DTT in a final reaction volume of 40 µl. Reactions were incubated for 90 min at 37°C at

220-240 rpms. Reactions were stopped and processed in one of two post-assay protocols.

Post-assay protocol 1: reactions were stopped with the addition of SDS-PAGE sample

buffer and boiled for 5 min. Samples were then separated via SDS-PAGE, followed by

transfer to polyvinylidene fluoride (PVDF) membrane. The membrane was then treated

with EA-Wax enhanced autoradiography reagent (EABiotech Ltd, Scotland, UK) as per


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manufacturer�s instructions and exposed to Biomax MS film at -80°C. Typical exposure

times were from 16 to 30 h.

Post-assay protocol 2: reactions were stopped by adding 0.5 ml TBST containing 0.5 M

NaCl. S-protein agarose beads were then added to the reactions to bind the S-OID

substrate. Beads were then washed 3 times with 1 ml of TBST (0.5 M NaCl). 10 ml of

scintillation fluid was added directly to the columns containing the beads, shaken briefly

and counted.

OGT peptide glycosylation assays were performed as described (27), except the

reaction buffer was 50 mM Tris pH 7.4, 12.5 mM MgCl2 and 1 mM DTT in a final

reaction volume of 40 µl. The CK II 12-mer peptide was used as a substrate at a final

concentration of 1 mM. All assays were performed in triplicate and each experiment was

performed at least two times.

Determination of Km of OGT for OID of OIP106: For the Km determination

experiments, assays were performed as described above with varying concentrations

(0.26 to 2.6 µM) of soluble S-OID protein substrate. 2 µg of OGT enzyme was used per

assay. The concentration of UDP-GlcNAc was kept constant at a final of 1 mM by cold

diluting from 34 Ci/mmol to 0.025 µCi/nmol with the addition of unlabeled UDP-

GlcNAc (Calbiochem). To remove the resulting UDP product which is a potent inhibitor

of OGT, 1 unit of calf intestinal phosphatase (NEB) was added to the reaction. Assays

were processed as per post-assay protocol 2. Units are defined as pmol of GlcNAc

transferred per µg of enzyme per min. All assays were performed under initial rate

conditions, in triplicate, and the Km was determined based on averaged results of two

independent experiments.


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TPR competition experiments: OGT protein and peptide assays in the presence of

competing TPR protein was performed as described above with the following parameters.

The S-OID protein (1.68 µM) and CK II peptide (55 µM) substrates were at a final

concentration of their respective Kms. His-TPR protein was added to the reaction at the

indicated amounts. Total protein amounts were kept constant with the addition of BSA.

Assays were processed as per post-assay protocol 2. Assays were performed in triplicate

and each experiment was performed twice.

OID-p62 glycosylation competition experiments: OID-p62 glycosylation competition

experiments were performed as follows. For p62 competition experiments, S-tagged OID

was kept at a constant concentration of 0.5 µM and the concentration of GST-p62 was

varied from 0.5 µM to 2.5 µM. Similarly, for OID competition experiments, GST-p62

was kept at a constant concentration of 0.5 µM and the concentration of OID was varied

from 0.5 µM to 2.5 µM. 425 ng of OGT was used per assay for each condition. In the 0

µM competitor control assays, 2.5 µM BSA was used to substitute the competing protein

substrate and serve as a negative control. Assays were performed at room temperature

for 90 minutes, as described in the above sections. Following the reactions, assays were

split into two and GST-p62 was purified by adding glutathione sepharose to one half and

the other half was subject to S-protein agarose chromatography to purify S-tagged OID.

Bound proteins from either affinity resin were eluted by boiling the resins in SDS sample

buffer (following extensive washing after adsorption). Glycosylation of p62 or OID were

analyzed by immunoblotting of the eluted proteins on PVDF, followed by analysis by the

O-GlcNAc specific monoclonal antibody CTD 110.6.


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Results

Localization of OIP106 and OGT Interaction Domains: Based on its strong interaction

with OGT, we hypothesized that OIP106 could function to influence OGT�s activity as a

possible co-factor (1). Therefore we first performed in vitro OGT assays to see if

recombinant OIP106 had any effect on OGT�s activity. Unfortunately, when

overexpressed in E.coli as a His-tagged fusion protein, the majority of recombinant full

length OIP106 was found to be insoluble. So we localized the region of OIP106 that was

responsible for its interaction with OGT, in hopes that this region would be a smaller

portion of the molecule and therefore be more amenable to overexpression in an E.coli

based system. In order to localize this region, we constructed N-terminal truncations of

OIP106. N-terminal OIP106 truncations were then synthesized in lysates as S-tagged

fusion proteins, pulled down with S-protein agarose and assayed for their ability to bind

endogenous OGT, as detected by anti-OGT (AL28) (1) western blotting. Figure 1 (top

panel) shows the various N-terminal OIP106 truncations synthesized as 35S-Met labeled

S-tagged fusion proteins. The bars to the right represent a graphical schematic of the

OIP106 truncations, lacking different domains. As seen in Figure 1 (bottom panel) , all

the truncations except the ∆859 (residues 860 to 953) truncation were able to bind and

pulldown OGT. This indicated that the potential OGT binding domain localized to

within residues 639 and 859 in the carboxy terminus of OIP106.

In order to confirm the above localization studies, we tested to see if the 220

amino acid fragment of OIP106 from residues 639 to 859 was sufficient to bind OGT.

We overexpressed this region in E.coli as a S-tagged fusion protein and purified it under

mildly denaturing conditions via S-protein affinity chromatography. We call this region


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OID for OGT Interacting Domain and performed in vitro binding assays with purified

recombinant OGT and the TPR domain. S-protein agarose alone or OID bound to S-tag

(S-OID), were incubated with purified baculovirus expressed recombinant OGT or the

TPR domain and S-protein pulldown assays were performed. As seen in Figure 2A, the

OID efficiently bound full length OGT (lane 3) and the TPR domain of OGT (lane 9), as

detected by anti-OGT (AL25) (20) western blot (top panel). S-protein agarose alone did

not bind any OGT or TPR domain (lanes 2 and 8, respectively). S-protein HRP blot

shows that equal amounts of S-OID were bound in each reaction (bottom panel). Thus,

the OID of OIP106 is sufficient to bind to OGT efficiently. These data confirm the

localization experiments performed above in lysates.

In order to further localize the specific TPRs that interact with the OID, we used

the ∆2.5 OGT and ∆5.5 OGT deletion mutants that lack the first 2.5 and 5.5 TPRs

respectively (33) in binding assays with S-tagged OID. As seen in Figure 2A (lanes 4-6),

the ∆5.5 OGT mutant fails to interact with the OID (lane 6). We performed binding

assays with the ∆2.5 OGT mutant and this is shown in Figure 2B. Binding assays were

performed under non-saturating conditions. Under physiological conditions (150 mM

NaCl), the ∆2.5 OGT is a trimer, whereas under high salt conditions (1 M NaCl), it forms

a dimer (33). ∆2.5 OGT-OID binding assays were performed under these two conditions.

As is seen in lanes 1-4, there were no significant differences in the binding of full length

OGT (Figure 2B, top panel) to OID (bottom panel) (compare lanes 3 and 4 of both

panels) between 0.15 M and 1 M NaCl binding conditions. Similarly, as is shown in

lanes 5-8 of Figure 2B, no significant differences were noticed in binding of the ∆2.5

OGT mutant (top panel) to OID (bottom panel) (compare lanes 7 and 8 of both panels) in


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either 0.15 M or 1 M NaCl binding condition. While the levels of ∆2.5 OGT appear be

diminished in the 1 M NaCl condition (lane 8), the levels of S-tagged OID protein that

was pulled down is also equally diminished (asterisk). We performed densitometry on

these bands and found that after normalization, the intensities of both ∆2.5 OGT and OID

bands in lanes 7 and 8 were similar. Therefore under physiological and high salt

conditions, the ∆2.5 OGT interacted with the OID as efficiently as the full length

wildtype enzyme, indicating that the OID (OIP106) can interact with either a dimeric or

trimeric form of OGT. Thus this implicates TPRs 2-6 of OGT as being the repeats that

interact with the OID of OIP106.

The OID of OIP106 is a High Affinity Substrate for OGT. We had previously

demonstrated that OIP106 is modified by O-GlcNAc in vivo (1). Furthermore, PROSITE

analysis of the OIP106 sequence revealed that the OID of OIP106 likely contains several

putative sites of glycosylation. In order to examine this, increasing amounts of OID were

incubated with OGT in in vitro protein glycosylation assays in the presence of UDP-[3H]-

GlcNAc. Reactions were stopped by adding SDS-PAGE sample buffer to the assays and

samples were separated by SDS-PAGE and subjected to autoradiography. The OID is

efficiently glycosylated by recombinant OGT, as seen in Figure 3B. Figure 3A

represents the Coomassie stained gel of the increasing amounts of S-OID used in the

glycosylation assay. The additional lower molecular weight bands observed in panel A

are proteolytic fragments of the S-OID fusion protein. OGT displayed standard

Michaelis-Menten kinetics when the OID was used as a substrate, as seen in Figure 3C

(left panel). The right panel shows the corresponding Lineweaker-Burk plot. The Km of

OGT for OID was determined to be 3.35 µM (+/- 1.45), which is very similar to the Km


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of nucleoporin p62 (1.2 µM) (13), one of the best known high affinity protein substrates

for OGT. On average, under optimal conditions, 2 moles of GlcNAc were transferred per

mole of OID by OGT in these assays.

The TPR Domain of OGT Functions as a �Docking� Site for Protein Substrates. It has

been shown previously that the TPR domain plays a role in trimerization of the OGT

(24). Other studies suggest that the TPR domain may be a site of substrate recognition

(13,24). But it is still unclear as to how the TPR domain may function in terms of protein

substrate recognition. We hypothesized that the TPR domain would form stable

complexes with its substrates in order for the OGT catalytic subunits to be able to

specifically glycosylate such a large diversity of proteins. In this model, the individual

TPRs confer substrate specificity by bridging the substrate to the enzyme. Thus based on

this model, we hypothesized that since the TPR deletion mutants are unable to efficiently

bind the OID of OIP106, that they would also be deficient in glycosylating the OID as

well. We assayed the ability of the ∆2.5 and ∆5.5 OGT mutants to glycosylate the OID

protein substrate, relative to their ability to glycosylate a twelve amino acid substrate

peptide based upon an O-GlcNAc site on the protein kinase CK II (24). These TPR

deletion mutants are able to efficiently glycosylate the CK II peptide (24). As is seen in

Figures 4D and 4E, both the ∆2.5 and ∆5.5 OGT mutants efficiently glycosylated the CK

II peptide, when compared to the full length OGT. This indicates to us that the purified

OGT mutant proteins are both catalytically active. However, both the ∆2.5 and the ∆5.5

OGT mutants showed a severely diminished capacity to glycosylate the OID protein

substrate, when compared with the full length enzyme. As seen in Figures 4A and 4B

(respectively), the ∆2.5 OGT is only 1/7th as active as the full length OGT towards the


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OID, whereas the ∆5.5 OGT completely fails to glycosylate the OID. It has been

previously shown that removal of the first 5.5 TPRs did not affect the OGT�s ability to

glycosylate peptide substrates (24). Yet removal of TPRs 1-6 severely affects the

capacity of the OGT to interact with and glycosylate the OID protein substrate. So while

removal of the first 2 TPRs does not affect binding of OGT to OID, they are clearly

important for efficient glycosylation of the OID of OIP106. Thus TPRs 1-6 of OGT are

necessary to efficiently interact with and glycosylate OIP106 (OID).

While these data are indicative of the role of the TPR domain in recognition of

protein substrates, we also performed TPR competition experiments to further document

the role of OGT�s TPRs in targeting substrates. The fact that the first 6 TPRs are

dispensable for activity towards peptide substrates led us to hypothesize that the TPR

domain should compete for the OID as a protein substrate with OGT, but should not

compete for the CK II peptide substrate. So OGT-OID protein or OGT-CK II peptide

glycosylation assays were performed in the presence of increasing molar amounts of TPR

domain. The molar ratio of TPR:OGT was kept constant in both the protein and peptide

assays. In order to keep the quantity of total protein constant, BSA was added to the

reactions. As is seen in Figure 4F (right panel), the TPR domain did not block CK II

peptide glycosylation, as expected. However, increasing amounts of TPR dramatically

reduce OID glycosylation by OGT (Figure 4C). A 12 fold molar excess of TPR

completely inhibits the glycosylation of OID. This indicates that the TPR domain is

competing with OGT for docking sites on the OID protein. These data suggest that one

of the functions of the TPR domain is indeed to serve as a binding/recognition site for

protein substrates. The observation that the TPR domain did not effect the glycosylation


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of the CK II peptide supports the observation that the catalytic site of OGT is in the

carboxy domain as earlier suggested by photolabeling analysis (13, 28).

Nucleoporin p62 and OID compete with each other for glycosylation by OGT. We have

shown that the OID of OIP106 is a high affinity substrate for OGT, similar to another

high affinity substrate, nucleoporin p62. However, p62 does not interact with OGT in a

stable manner, such as OIP106 (1). In order to examine possible differences in the

specificity of these two high affinity substrates vis-à-vis OGT, we performed

glycosylation assays on each substrate, in increasing concentrations of the other substrate

to study possible competition effects. OGT assays were performed using constant

amounts of recombinant GST-p62 (0.5 µM), in the presence of increasing concentrations

of OID (0.5 µM to 2.5 µM). Similarly, assays were performed using constant amounts of

OID (0.5 µM), in the presence of increasing concentrations of GST-p62 (0.5 µM to 2.5

µM). Competition of each substrate for OGT with each other was examined by analyzing

the glycosylation of each substrate via the O-GlcNAc specific monoclonal antibody CTD

110.6 (39). As is seen in Figure 5 (top panel), glycosylation of OID was effectively

competed away by increasing amounts of GST-p62. Increasing presence of GST-p62

resulted in increased glycosylation of GST-p62 by OGT and a corresponding decrease in

the glycosylation of OID. The control GST alone protein was not glycosylated by OGT

(data not shown). In addition, 2.5 µM BSA was used in the 0 µM competitor control

assays (lanes 1 and 5) to keep the protein quantities constant and serve as a negative

control; therefore the effects of competition observed are not due to a protein mass action

effect. Similarly, as seen in Figure 5 (bottom panel), glycosylation of GST-p62 was

effectively competed away by increasing amounts of OID. Increasing concentrations of


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OID resulted in increased glycosylation of OID by OGT and correspondingly, a decrease

in the glycosylation of GST-p62 in an OID dose dependant manner. In both cases, a 5

fold molar excess of competitor to substrate was sufficient to significantly compete away

glycosylation. It should be noted that while the 5 fold molar excess of OID completely

inhibited p62 glycosylation (lane 8, bottom panel), the same molar excess (5 fold) of

GST-p62 did not completely inhibit OID glycosylation, as some residual glycosylation is

still observed (lane 4, top panel). It is possible that higher molar amounts of GST-p62

may be required to completely inhibit OID glycosylation. While it appears that the level

of inhibition of p62 glycosylation by OID appears to be similar in all concentrations

tested, competition of p62 glycosylation was OID concentration dependant in other

similar assays that were performed (data not shown) and therefore the lack of OID

concentration dependant inhibition is an artifact of this particular experiment, shown in

Figure 5. Thus both high affinity substrates compete with each other for glycosylation by

OGT.


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Discussion

Mediation of OGT protein substrate specificity via its TPR domain:

Previous work on OGT enzymology have clearly shown the requirement of its

TPR domain in modulating its oligomerization, as well as specificity towards its peptide

substrates in vitro (24). A report by Lubas et. al (13) have shown that deletion of TPR

repeats affects OGT�s activity towards a nucleoporin p62 protein substrate. Our studies

contribute to new knowledge about OGT in four major aspects. First, Lubas et al (13)

had performed their studies with a truncated form of recombinant OGT lacking the first

two and a half TPRs. This group had previously inadvertantly cloned a partial clone of

human OGT which only contained 9 TPRs (21). Subsequently, Nolte and Muller (29)

have shown that the human OGT gene indeed does, in fact, contain the missing two and a

half TPRs. More recently, it was shown that the truncated form of OGT containing only

9 TPRs contains a mitochondrial targeting sequence in its amino terminus and targets to

the mitochondria (40). Furthermore, this form of OGT, termed mOGT by Love et al.,

was not active toward mitochondrial protein substrates, suggesting a different function

for this form of the enzyme (40). Therefore, enzymatic studies performed with this

truncated form of OGT are not conclusive, since the wildtype nucleocytoplasmic form of

OGT (containing its entire complement of TPRs) was not assayed and compared to with

the mutants used in these experiments. In our report, we performed our studies with the

correct wildtype nucleocytoplasmic isoform of OGT and its truncation mutant enzymes.

Furthermore, we characterize the glycosylation of an OGT substrate that is novel i.e the

OID of OIP106. The second major findings are the TPR competition data (Figure 4C and

F). By virtue of the fact that free TPR domain inhibits OID glycosylation by OGT and


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competes for its binding, but not for the CK II peptide substrate, clearly indicates one of

its roles as a protein substrate �docking/binding� domain. These data indicate that free

TPR is not interfering with the OGT holoenzyme�s catalytic activity in a mass action

effect, based on its inability to compete away CK II peptide glycosylation. Our finding

that the TPR domain did not compete for the CKII peptide substrate also provides

evidence for the notion that peptide substrates weakly interact (KM CK II peptide = 103

uM) (24) with the catalytic site at the carboxy terminus of OGT. These data also further

support the model that OGT has two distinct modular halves, with the carboxy half of the

enzyme containing the putative catalytic domain and the amino TPR half regulating its

activity by docking to specific proteins.

The third contribution is the finding that OGT forms stable complexes with the

OID (Figure 2) in a TPR dependant manner and this TPR dependence is mirrored by its

glycosylation of OID. This brings up the issue of the multimerization of the enzyme, as it

has been previously shown the TPR domain modulates the OGT's ability to associate

with itself (24). Our data states that multimerization of the enzyme is not sufficient for

its activity toward the OID protein substrate, as the ∆2.5 OGT mutant (which is a dimer

at 1 M NaCl) binds to the OID as efficiently as the wildtype full length OGT (Figure 2B)

and therefore the OID can interact either with the dimeric or trimeric form of OGT. The

∆5.5 OGT mutant is a monomer (24) and thus the monomeric form of OGT neither

interacts nor glycosylates the OID protein substrate. However, the ∆2.5 OGT is severly

diminished in its capability to glycosylate the OID, even though it is fully active toward

the CK II peptide substrate (Figure 4A and D). It should be noted that the ∆2.5 OGT is a

trimer under assay conditions, where no salt is present (as even low amounts of NaCl


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potently inhibits enzyme activity, 27). Thus toward a unique protein substrate such as

OIP106 (OID), a stable association as well as atleast dimerization of the enzyme are pre-

requisites for activity, since loss of another 3 TPRs results in loss in binding as well as

complete loss of activity (∆5.5 OGT-OID binding and glycosylation assays, Figure 2A

and Figure 4B, E respectively). This may be a way of how the TPRs (specifically

involving the first 2.5 TPRs) govern the OGT's specificity toward an stably interacting

protein substrate such as OIP106 (and possibly GRIF-1), possibly by regulating the

enzyme's multimerization status. However, it is currently unknown if the multimer-

ization of OGT changes in response to cellular stimuli or events and remains to be

studied. It can be speculated that perhaps levels of UDP-GlcNAc or other factors in the

cell may modulate the enzyme's multimerization status, since it is known that the OGT is

highly responsive to even slight changes in cellular UDP-GlcNAc concentrations (41).

Interestingly, we found that the the OID of OIP106 stays tightly associated with

the enzyme, even after it is glycosylated. S-protein pulldown experiments performed on

OID glycosylation reactions prior to and after glycosylation showed no difference in the

amount of OGT that was retained by S-tagged OID (data not shown). Similarly, OGT

stays bound to the OID in saturating concentrations of UDP-GlcNAc, at concentrations as

high as 3.43 mM (data not shown). Thus, the OGT remains associated with the OID

tightly, even after it is glycosylated. We have shown previously that nucleoporin p62,

which has a similar Km for OGT, does not form such stable complexes with OGT,

whereas OIP106 forms stable in vivo and in vitro complexes with OGT (1). In the fourth

major contribution of this paper, we sought to explore the fundamental differences

between the two substrates and their respective behavior toward OGT in a variety of


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experiments. We hypothesized that perhaps nucleoporin p62 (a "non-interacting

substrate") would be unable to compete away OID glycosylation by OGT, whereas the

OID of OIP106 would be able to compete away p62 glycosylation by OGT, by virtue of

being an "interacting substrate". However, unexpectedly, both substrates competed

effectively with each other for glycosylation by OGT (Figure 5). We then analyzed

enzyme-substrate reactions (OID-OGT and p62-OGT) via Superdex 200 gel filtration

chromatography, prior to and after glycosylation. We expected to find possible changes

in the structure of the enzyme-substrate complexes, as would have been indicated by

differences in migration patterns, and thus clues to the differences between the two

substrates. However, we could not find any significant discernable differences in these

complexes (data not shown). It is possible that the differences between the two substrates

and how OGT interacts with them, lies in the individual sets of TPRs that interact with

them. The set of TPRs that interact (transiently) with p62 may specify transient substrate

interactions ("non-interacting substrates"), whereas the TPRs that interact with OIP106

may dictate more stable associations ("interacting substrates"). Discerning the individual

sets of TPRs that interact with either substrate (via limited proteolysis or cross-linking

experiments) may help explain these fundamental differences.

Given the above data, we suggest a model for targeting of the OGT by binding to

the OID of OIP106. It can be envisioned that once glycosylated, OIP106 then can target

its associated OGT (stoichiometrically) to distinct subcellular locations in the cell. These

distinct locations could be the speckle or dot-like regions that OIP106 localizes to in the

nucleus (1). We have shown that OIP106 exists in a complex with RNA pol II and OGT

(1). Thus the TPRs 1-6 would mediate this targeting of OGT to transcriptional


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complexes. A similar targeting mechanism can be suggested for GRIF-1, in context of

the possible targeting of OGT to GABAA receptor complexes.

Individual TPRs that have specific functions have been well documented in the

literature, especially in the case of Ssn6, where specific sets (tandem and non-tandem) of

its TPRs mediate specific protein-protein interactions with different binding partners

(23). Similar interactions have also been observed within the yeast cell cycle control

proteins Cdc16, Cdc23 and Cdc27. These proteins contain TPR domains and interact

with each other via their TPR domains, and each specific interaction is mediated by

different TPR combinations (30). The crystal structure of the TPR domains of protein

phosphatase 5 (PP5) (31) and Pex5p (32) have been solved. Each repeat of the 3 TPRs of

PP5 have been shown to form pairs of anti-parallel alpha helices (31). A 12 TPR protein

modeled after the PP5 TPR crystal structure indicates that TPRs arranged in tandem, such

as the eleven and a half TPRs of OGT, would be organized into a regular right-handed

super-helix (31). This type of arrangement would facilitate multiple interactions with

multiple proteins, using specific combinations of TPR motifs within the super helix.

Recently, a report by Yang et.al (25) showed that OGT is present in a transcriptional

repressor complex with corepressor mSin3A. Interestingly, the first 6 TPRs of OGT

were implicated in interaction of OGT with this complex. Since OIP106-OGT

interactions also involve the first 6 TPRs, pinpointing the exact sets of TPRs that mediate

the interactions between the two proteins should help in delineating the specificity

between OIP106-Pol II-OGT and OGT-mSin3A complexes.


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28. Kreppel, L. K. (1999) Cloning and Characterization of a Unique Nuclear and

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Acknowledgments:

We would like to thank Dr. Zhiyu Li for providing the recombinant OGT and the

TPR deletion mutants. This work was supported by NIH HD13563 to G. W. H. Under a

licensing agreement between Covance Research Products, Hoffman LaRoche, and The

Johns Hopkins University, Dr. Hart receives a share of royalty received by the university

on sales of the CTD 110.6 antibody. The terms of this arrangement are being managed by

The Johns Hopkins University in accordance with its conflict of interest policies.

Footnotes:

The abbreviations used are: O-GlcNAc, β-O-linked N-acetylglucosamine; OGT, uridine

diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase;

O-GlcNAcase, N-acetyl-β-D-glucosaminidase; GlcNAc, N-acetylglucosamine; TPR,

tetratricopeptide repeat; OIP106, OGT Interacting Protein of 106 kDa; CC, coiled-coil

domain; HBS, Hepes buffered saline; TBS, Tris buffer saline; PBS, phosphate buffered

saline; PMSF, phenylmethylsulfonyl fluoride; amp, ampicillin; HRP, horseradish

peroxidase; IMAC, immobilized metal affinity chromatography; GRIF-1, GABAA

receptor associated protein; OID, OGT interacting domain (of OIP106); GST,

glutathione-S-transferase; p62, nucleoporin p62.


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Figure Legends

Figure 1: Localization of the OGT interaction domain (OID) of OIP106. (A) N-

terminal deletions of OIP106 were generated as S-tagged constructs and were

synthesized in vitro in rabbit reticulocyte lysates (top panel). S-tagged proteins were

purified via S-protein affinity chromatography and bound proteins were eluted with SDS-

PAGE sample buffer, boiled and analyzed via autoradiography (top panel) and anti-OGT

(bottom panel). The bars to the right represent the N-terminal truncations. The black bar

represents the coiled-coil domains and the shaded bar represents the OID. The diamond

in front of each of the OIP106 constructs represents the position of the amino terminal S-

tag. nCC = amino terminal coiled coil domains (CC1 + CC2). The numbers to the left

represent molecular weight markers.

Figure 2: The OID of OIP106 efficiently binds to full length OGT and its TPR

domain, but not to the ∆ 5.5 OGT deletion, lacking the first five and a half TPR

domains. (A) Recombinant full length OGT, the ∆ 5.5 OGT deletion and the TPR

domain were incubated with recombinant S-tagged OID and pulled down with S-protein

agarose. Protein complexes were washed extensively and bound proteins were eluted

with SDS-PAGE sample buffer and analyzed with anti-OGT antibodies (top panel) and

S-protein HRP (bottom panel). Lanes 1-3 show the full length OGT-OID binding assays,

whereas lanes 4-6 represent the ∆5.5 OGT-OID binding assays. Lanes 7-9 show the TPR

domain-OGT binding assays.

(B) ∆2.5 OGT interacts with the OID of OIP106 similarly as wildtype OGT, under

phsiological and high salt conditions. ∆2.5 OGT-OID binding assays were performed

under physiological salt (0.15 M NaCl) (trimer) or high salt (1 M NaCl) (dimer)


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conditions. Similarly, wildtype OGT-OID binding assays were performed in parallel as a

control. Assays were performed under non-saturating conditions. As is seen in lanes 3

and 4, no significant differences were seen in the binding of wildtype full length OGT to

OID either under physiological (0.15 M NaCl, lane 3) or high salt (1 M NaCl, lane 4).

Similarly, as is shown in lanes 7 and 8, no significant differences were seen in the

binding of the ∆2.5 OGT (top panel) to OID under either 0.15 M (lane 7) or 1 M NaCl

(lane 8) salt conditions. While it appears that levels of ∆2.5 OGT are reduced in the 1 M

NaCl pulldown (lane 8), the S-protein HRP blot on the bottom panel shows the amount of

S-OID pulled down by S-protein agarose in lane 8 is less than that in lane 7 (asterisk).

However, when normalized (densitometry), the levels of both proteins (OID and ∆2.5

OGT) in both lanes are similar, indicating that there are no differences in binding of ∆2.5

OGT to OID in either salt condition.

Figure 3: The OID of OIP106 is a high affinity substrate for OGT. (A) Recombinant

S-tagged OID was overexpressed and purified via IMAC. Panel A represents increasing

amounts of purified OID that was used as a substrate for OGT in in vitro OGT protein

assays. (B) OGT glycosylates OID in a dose dependant manner. Panel B represents a

27 h autoradiograph of the OID glycosylation assay. (C) The OID is a high affinity

substrate of OGT and has a Km similar to other high affinity protein substrates.

OGT protein assays were performed as described, using increasing amounts of OID (0.48

to 2.6 µM) and saturating amounts of UDP-[3H] -GlcNAc, supplemented with 1 mM

unlabeled UDP-GlcNAc. Labeled OID was purified with S-protein affinity

chromatography, washed with TBST containing 0.5 M NaCl and counted. The left panel

represents the Michaelis-Menten kinetics of OID glycosylation and the right panel


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represents the corresponding Lineweaver-Burk plot. A Km of 3.35 µM was determined

as an average of two independent experiments.

Figure 4: ∆2.5 OGT trimer is partially towards OID protein substrate, but is fully

active towards the CK II peptide substrate. OGT assays were performed as described,

using either (A) recombinant OID protein (left panel) or (D) CK II peptide (right panel)

as substrates. Source of enzyme was recombinant ∆2.5 OGT purified as described

before. ∆5.5 OGT monomer is inactive towards OID protein substrate, but is fully

active towards the CK II peptide substrate. OGT assays were performed as described,

using either (B) recombinant OID protein (left panel) or (E) CK II peptide (right panel)

as substrates. Source of enzyme was recombinant ∆5.5 OGT , purified as described

before. TPR domain of OGT competes for binding of OID with OGT, but not for

binding to CK II peptide. OGT assays were performed as described, with either (C)

OID protein substrate (1.68 µM) (left panel) or (F) CK II peptide substrate (55 µM) (right

panel), in the presence of increasing molar excess of TPR to OID.

Figure 5: Nucleoporin p62 and OID of OIP106 compete with each other for

glycosylation by OGT. OGT assays were performed using the S-tagged OID or GST-

p62 protein substrate at a constant concentration of 0.5 µM, while the concentration of

GST-p62 or S-OID protein competitor was varied from 0 µM to 2.5 µM. Following

assays, S-OID and GST-p62 proteins were purified via S-protein and glutathione affinity

chromatography and their levels of O-GlcNAcylation analyzed by anti-O-GlcNAc

monoclonal antibody CTD 110.6 (top and bottom panels, respectively). In lanes 1-4, S-

OID was kept constant at 0.5 µM, whereas GST-p62 competitor was varied from 0-2.5

µM. In lanes 5-8, GST-p62 was kept constant at 0.5 µM, whereas S-OID competitor was


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varied from 0-2.5 µM. In assays where no competitor was present (i.e 0 µM competitor;

lanes 1 and 5), 2.5 µM BSA was substituted to keep the protein concentrations constant.


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Autoradiograph

____

_

_

_

_

_

178111796149

36

24

19

13

Vector Full ∆nCC ∆491 ∆639 ∆859Anti-OGT

OGT

OIP106 N-terminal truncations schematic

Full length

∆nCC

∆491

∆639

∆859

nCC CC3

OID

OID

OID

OID

= S-tag

Figure 1

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FulllengthOGT

178

1117962

OGT con OID ∆ 5.5 con OID TPR Con OIDinput PD PD input PD PD input PD PD

Lane # 1 2 3 4 5 6 7 8 9Anti-OGT

∆5.5 OGT

TPR

49

36

49S-OID

36S-proteinHRP blot

Figure 2A


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OGT (-) 0.15 1 M ∆2.5 (-) 0.15 1 MInput PD OID input PD OID

∆2.5 OGT

S-OID

Lane 1 2 3 4 5 6 7 8

*

Anti-OGT

FulllengthOGT

S-protein HRP blot

Figure 2B


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Figure 3

C.

Km = 3.35 µµµµM ± 1.45

r2 = 0.993

0.24 0.48 1.2 2.4 4.8 µµµµg S-OID0.13 0.26 0.65 1.3 2.6 [OID] µµµµM

A. B.

OID

AutoradCoomassie G-250 stain178

111

79

62

49

36

0 0.24 0.48 1.2 2.4 µµµµg S-OID0 0.13 0.26 0.65 1.3 [OID] µµµµM

v (p

mol

/ug/

min

)

0

0.010.020.03

0.04

0.050.06

0.07

0 0.5 1 1.5 2 2.5 3[OID] µµµµM

0

25

50

75

100

125

0 1 2 3 41/[OID]

1/v


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No No Full length ∆∆∆∆2.5 OGTenzyme OID OGT

No No Full length ∆∆∆∆2.5 OGTenzyme CKII OGT

DPM

s/ug

OG

T a

ctiv

ity (

x 10

5 )

OID protein substrate CK II peptide substrate

0

2

4

6

8

10

12

14

16

18

0

1

2

3

4

5

DPM

s/ug

OG

T a

ctiv

ity (

x 10

3 )

A. D.

Figure 4


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No enzyme No OID Full length ∆∆∆∆5.5 OGTOGT

OID protein substrate

No enzyme No CKII Full length ∆∆∆∆5.5 OGTOGT

CK II peptide substrate

DPM

s/ug

OG

T a

ctiv

ity (

x 10

3 )

0

5

10

15

20

25

30

35

40

DPM

s/ug

OG

T a

ctiv

ity (

x 10

5 )

0

1

2

3

4

5B. E.

Figure 4 (cont)


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DPM

s/ug

OG

T a

ctiv

ity (

x 10

4 )

No OID OGT 1:1 1:5 1:12 OID alone + (-) BSA OID + TPR

(OID:TPR molar ratio)

No CKII CKIICKII alone +

(-) BSA

OID protein substrate CK II peptide substrate

1.8x 39.3x 94.2x (molar excess of TPR to OGT)

1.8x 39.3x 94.2x (molar excess of TPR to OGT)

TPR

DPM

s/ug

OG

T a

ctiv

ity (

x 10

3 )

5

0

10

15

20

25

30

35

2

0

4

6

8

10C. F.

Figure 4 (cont)


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OGT + OIDuM p62

0 0.5 1.25 2.5

OID

0 0.5 1.25 2.5

OGT + p62uM OID

Anti-O-GlcNAc(CTD 110.6)

p62

Lane 1 2 3 4 5 6 7 8

Figure 5


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OID

p62

0 0.1 1 mM [CKII peptide]

Anti-O-GlcNAc blot (CTD 110.6)

0 200 2000 fold CKII excess

p62 and OID at constant concentration of 0.5 uM


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Sai Prasad N. Iyer and Gerald W. Hartsubstrate specificity

Roles of the TPR domain in O-GlcNAc transferase (OGT) Targeting and protein

published online April 30, 2003J. Biol. Chem.

10.1074/jbc.M300036200Access the most updated version of this article at doi:

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Documents

Roles of the TPR domain in O-GlcNAc Transferase …cells (20,21). The OGT gene resides on the X chromosome near the centromere (Xq13 in man), and its targeted deletion results in embryonic