From the Center for Oral Biology, Rochester Institute
of Biomedical Sciences, University of Rochester, Rochester, New York
14642 and the § Department of Medical Microbiology and
Immunology, University of Alberta, 1-41 Medical Sciences Building,
Edmonton, Alberta T6G 2H7, Canada
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ABSTRACT |
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Mucin-type O-glycosylation is
initiated by a family of UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferases (ppGaNTases). Based on
sequence relationships with divergent proteins, the ppGaNTases can be
subdivided into three putative domains: each putative domain contains a
characteristic sequence motif. The 112-amino acid
glycosyltransferase 1 (GT1) motif
represents the first half of the catalytic unit and contains a short
aspartate-any residue-histidine (DXH) or aspartate-any
residue-aspartate (DXD)-like sequence. Secondary structure
predictions and structural threading suggest that the GT1 motif forms a
5-stranded parallel UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferases (EC 2.4.1.41;
ppGaNTases)1 comprise a large
family of metal-dependent enzymes that initiate mucin-type
O-glycosylation. During catalysis, GalNAc is transferred from UDP-GalNAc to selected serine and threonine residues of proteins destined for the extracellular environment. The family of ppGaNTases have been conserved during evolution (1). Since
O-glycosylation proceeds in a stepwise manner, the
expression and specificity of ppGaNTase isozymes represent key
regulatory factors in defining the repertoire of O-glycans
expressed by a cell. In vitro studies have shown that some
ppGaNTases (ppGaNTase-T1, -T2, and -T3) glycosylate a broad spectrum of
peptides (2, 3), whereas others exhibit more restricted (ppGaNTase-T4
and -T5) or unique (ppGaNTase-T3) substrate preferences (2, 4, 5). The
structural basis for this enzyme specificity is unknown, as no
experimental structure is currently available for ppGaNTases or for any
other mammalian glycosyltransferase.
Recently, several groups have identified an aspartate-any
residue-aspartate (DXD) (or aspartate-any residue-histidine
(DXH)) sequence motif that is common to many
glycosyltransferases (6-9). All known ppGaNTases contain such a
DXH or DXD-like motif. Based on fold recognition,
the DXD motif of the In the current study, threading analysis suggests that the central
340-amino acid region of mammalian and Caenorhabditis
elegans ppGaNTases share a common structural fold that consists of
two domains, each of which contains a parallel Secondary Structure Predictions and Threading Analysis--
The
multiple sequence alignments of ppGaNTases were prepared with ClustalW
(17). The secondary structure prediction was created by the JPRED
server (http://circinus.ebi.ac.uk:8081/) using all sequences listed in
Fig. 2A plus the other closely related ppGaNTase isoforms.
The sequence homology between the ppGaNTase and
Accession Numbers for ppGaNTases Sequences--
The sequences
which include bovine, human, murine, porcine, and rat-T1 amino acid
sequences are >98% identical and are available with accession numbers
539755, 2135942, 2149049, 1339955, and 1709559. Human-T2 is 2135941. Human and murine-T3 are 1617312 and 1575723. Murine-T4 is 2121220;
rat-T5 is 3510639. C. elegans ppGaNTase sequence homologs
GLY3, GLY4, GLY5a, GLY6a, GLY7, GLY8, GLY9 are AF031833, AF031834,
AF031835, AF031838, AF031841, AF031842, AF031843, respectively.
Full-length GLY10, GLY11 cDNA sequence is not
complete.2
Accession Numbers for Plasmid Constructs and Mutagenesis--
Mouse ppGaNTase-T1
isoform was amplified and cloned from a first-strand cDNA synthesis
reaction of mouse kidney total RNA, using the following sense and
antisense oligonucleotide primers Mlu-mT1
(CACACGCGTTGCCTGCTGGTGACGTTCTAGAGCTAGT) and Bam-mT1
(ATGCGGATCCAGCCCAGTCAATCCTTCCTT), respectively. Final cDNA sequence
was obtained by sequencing both strands of multiple polymerase chain
reaction-derived clones. The cDNA was cloned into a M13 vehicle and
uracil-containing single-stranded DNA was prepared in the
Escherichia coli strain CJ236. Mutagenesis, according to the
procedure described by Kunkel (20), was first used to create a
MluI cloning site in the cDNA encoding the stem region
of the enzyme at amino acid 40, which is the position of the native N
terminus for the secreted form of the enzyme. This construct,
designated M13-mT1-Mlu, was used as a uracil-containing parent vector
for all mutagenesis experiments. Mutants were identified by restriction
enzyme screening and verified by DNA sequencing using a LiCOR 4000 DNA
sequencer. Positive clones were subcloned into the COS7 cell expression
vector pIMKF1, which contains an insulin secretion signal and a FLAG
antibody recognition sequence (4). All expression constructs with
mutants were verified by DNA sequencing. The initial mutations
introduced BssHII cloning sites in the stem region at amino
acid codon positions 56 and 73, which allowed for the construction and
expression of truncated recombinant enzymes. While the Expression and Immunopurification of Recombinant
Proteins--
Recombinant ppGaNTases were expressed as secreted
products from COS7 cells and partially purified from cell culture
medium as described previously (4). To normalize the quantity of
recombinant proteins employed for enzyme assays, aliquots of the
immuno-purified enzymes were 32P-labeled with heart muscle
kinase and separated by SDS-PAGE, and the level of product was
estimated by PhosphorImager analysis (Molecular Dynamics) as described
previously (5). Unless noted, expression levels of all single point
mutants of ppGaNTase-T1 were within the same order of magnitude of
wild-type levels, and activities appeared to be stable during the time
course of the in vitro glycosylation assays.
ppGaNTase Assays--
Enzyme activity was measured by
quantifying the transfer of [14C]GalNAc to the peptide
acceptor EA2 (PTTDSTTPAPTTK) using conditions previously described (4).
Apparent Km values were obtained for UDP-GalNAc,
using a nucleotide-sugar concentration series ranging from 2.5 to 80 µM with the EA2 peptide held at 500 µM. The
apparent Km measurements for EA2 peptide used a
peptide concentration series ranging from 31.25-1000 µM with a final concentration of 50 µM UDP-GalNAc. All
transfections and enzyme assays were performed in duplicate and
repeated twice.
General Structural Features of the ppGaNTases--
In common with
other glycosyltransferases that localize to the Golgi complex, the
ppGaNTases are type II membrane proteins, characterized by a short
(4-24 aa) N-terminal cytoplasmic tail, followed by a small (15-25 aa)
transmembrane anchor, which is tethered to a large (>450 aa) segment
in the lumen of the Golgi by a stem region of variable length. In Fig.
1A, the amino acid sequence of
murine ppGaNTase-T1 is presented together with the structural features
described above and the sequence motifs and structural elements to be
described in the subsequent sections.
To define the catalytic unit of the ppGaNTase-T1, three approaches were
taken: 1) putative domains of potential functional importance were
delineated by identifying ppGaNTase sequence motifs that were related
to motifs present in distant protein families (detected by either
PSI-BLAST or Hidden Markov Modeling); 2) secondary structure
predictions and fold recognition was used to propose a structural model
for each motif (using JPRED and THREADER); and 3) mutagenesis was used
to test the role of the conserved features of each motif in enzyme
function. In this manner, three putative structural domains were
identified in the lumenal regions of ppGaNTases and the boundaries of
the stem were defined (Fig. 1B). The putative stem regions
of ppGaNTases are diverse both in terms of composition and length,
ranging from 55 to 416 aa for ppGaNTase-T1 and -T5, respectively.
Proteolytic cleavage within the stem region of many type II membrane
proteins leads to the release of a soluble circulating species. From
amino acid sequencing, it is known that the ppGaNTase-T1 is cleaved at
residue 40 (21 and Fig. 1A).
In contrast to the highly variable stem region, all known ppGaNTases
share a highly conserved block of approximately 340 aa, which span
about two-thirds of the lumenal portion of the enzyme (Fig. 1,
A and B). Contained within this block of 340 aa
are two unique sequence motifs. The first sequence motif is common to a
wide range of
glycosyltransferases3 and
hence termed the glycosyltransferase
1 (GT1) motif. The second sequence motif is found in
Identification of Essential Residues within the GT1 Motif--
The
GT1 motif was identified by PSI-BLAST and Hidden Markov
Modeling.3 This motif spans 112 amino acids, beginning at
position 117 and ending at position 229 in ppGaNTase-T1 (Fig. 1 and
2A). Secondary structure
predictions of the GT1 motif indicate that the sequence is arranged as
a series of alternating
ppGaNTases, which are retaining glycosyltransferases, are thought to
work via a double-displacement mechanism involving at least two
carboxylic acid residues (12). Amino acid sequence alignment of this
region reveals 4 invariant carboxylic acid residues and 1 invariant
histidine. Conservative mutagenesis of any one of the 4 invariant
aspartates and glutamates in the GT1 motif to their amide forms
resulted in a >99.8% loss of enzyme activity (Fig.
3). This indicates that a carboxylate is
crucial at 4 specific sites: E127Q, D156Q, D209N, and E213Q. A D156N
mutant was not made because this would have created a potential
N-glycosylation site. Aspartate 209 was also mutated to an
alanine and glutamate (D209A and D209E) to examine the effect of
changing the size and charge of this essential residue. Mutants D209N
and D209A, while stably expressed, had no detectable enzyme activity
(<0.04%), whereas D209E exhibited a very low but significant level of
activity (0.29%) relative to wild type. This latter result suggests
that the negative charge of the aspartate carboxyl group is absolutely necessary for catalytic activity and that its spatial position is
extremely important. As a control, we mutated two carboxylate side
chains that were highly conserved (but not invariant) to their amide
forms (E150Q and D155N). Both mutants exhibited a wild type level of
enzyme activity. In the case of D155N, the specific activity of the
enzyme was not significantly affected; however, the expression of
recombinant protein from COS7 cells was markedly compromised (Fig. 3).
To confirm that this low level of protein production was not due to a
cloning artifact, four independent mutants were constructed, sequenced,
and expressed in COS7 cells. In each case, the D155N mutation resulted
in a low level of protein expression, suggesting that the preference for aspartate at position 155 of the GT1 motif serves a structural rather than functional role.
Sequence comparison indicates that the most highly conserved position
in the GT1 motif corresponds to aspartate 209 in ppGaNTase-T1. The
aspartate 209 analogue in three other GT1-containing
glycosyltransferases (chitin synthase 2, MNN1
Previously, we had reported that a H125A mutation inactivates
recombinant bovine ppGaNTase-T1 (24). With a larger data set of
ppGaNTases, it is now clear that this position, albeit highly conserved
(found in 10 out of 14 ppGaNTase isoforms), is not invariant. Both the
H125Q and H125F mutants were active, with the H125F mutant having a
near 3-fold greater activity than wild type (Fig. 3). These results
suggest that an aromatic group at position 125 enhances enzyme
activity. Interestingly, a phenylalanine or tyrosine is preferred at
this position in the GT1 motif of many other glycosyltransferases, suggesting that it serves a function common to the
family.3
Identification of Essential Residues within the Gal/GalNAc-T
Motif--
The Gal/GalNAc-T sequence motif (Fig. 1) was identified by
PSI-BLAST analysis, which revealed a 41-aa segment that has a distant sequence similarity to the
As a control, four highly conserved but not invariant residues have
been mutated (N320A, D375A, D375N, E376Q, H341A, H341L, H341V, H341K,
and H341R), but these mutations had little effect on enzymatic activity
(Fig. 4C). With the accumulation of a larger sequence data
base of ppGaNTase isoforms (1-5), it is now apparent that histidine
341 is not as conserved as previously thought (24). In contrast,
histidine 344, is invariant among all known ppGaNTases and is essential
for catalytic activity (24).
The Ricin-like Sequence of ppGaNTases Is Not Essential for
Catalytic Activity--
The presence of a ricin-like sequence in the C
terminus of ppGaNTase-T1 raised the possibility that this region may
represent the binding site for the sugar donor UDP-GalNAc or for
partially glycosylated peptide substrates (22, 23). In ClustalW
alignments of the first of three repeats in the ricin-like motif, it is
apparent that the key structural elements and carbohydrate-binding
residues of ricin are preserved in the C-terminal end of most but not
all ppGaNTases (Fig. 5A). For
murine ppGaNTase-T1, Asp-444 and Asn-465 model to the conserved
positions of the Essential Amino Acid Residues of ppGaNTases Lie Near the Predicted
C-terminal Ends of
The threading analysis with the second ppGaNTase domain revealed a weak
match to the second domain of the lac repressor. Essential glutamates
of the WGGENXE sequence motif lie in the active site cleft
at the predicted C-terminal end of strand
The lac repressor structure model suggests that both the first and
second domains of the catalytic unit in ppGaNTases contribute critical
residues for the binding of the sugar donor, sugar acceptor, and
manganese ion. To determine if binding affinities for the sugar donor
and acceptor could be dissociated from one another, kinetic parameters
were measured for UDP-GalNAc and the EA2 peptide for ppGaNTase-T1
mutants that retained enzyme activity. For each mutant tested, the
Km values for both UDP-GalNAc and peptide acceptor
were simultaneously affected by a single mutation, suggesting an
interdependence of binding of both substrates (data not shown). The
lactose repressor and the bacteriophage T4 DNA
In the absence of a definitive experimental structure, our data provide
a starting point for the creation of ppGaNTase-specific inhibitiors and
mutagenesis studies to both enhance ppGaNTase activity and modify
substrate specificity. Studies of this type are currently in progress.
-sheet flanked by 4
-helices, which resembles
the first domain of the lactose repressor. Four invariant carboxylates
and a histidine residue are predicted to lie at the C-terminal end of
three
-strands and line the active site cleft. Site-directed
mutagenesis of murine ppGaNTase-T1 reveals that conservative mutations
at these 5 positions result in products with no detectable enzyme
activity (D156Q, D209N, and H211D) or <1% activity (E127Q and E213Q).
The second half of the catalytic unit contains a
DXXXXXWGGENXE motif (positions 310-322) which is also found in
1,4-galactosyltransferases (termed the Gal/GalNAc-T motif). Mutants of carboxylates within this motif express either no
detectable activity, 1% or 2% activity (E319Q, E322Q, and D310N, respectively). Mutagenesis of highly conserved (but not invariant) carboxylates produces only modest alterations in enzyme activity. Mutations in the C-terminal 128-amino acid ricin-like lectin motif do
not alter the enzyme's catalytic properties.
INTRODUCTION
Top
Abstract
Introduction
References
1,3-fucosyltransferase is predicted
to correspond to aspartate 100 in the active site of the bacteriophage
T4 DNA
-glucosyltransferase (7, 10). Mutagenesis of aspartate
residues in other DXD-containing enzymes (mannosyltransferase, glucosyltransferase, and chitin synthase 2)
demonstrates that they are essential to catalytic activity (7, 9, 11).
Such carboxylic acid residues may be involved in different aspects of
the catalytic process. First, glycosyltransferases (including
ppGaNTases) that retain the anomeric configuration of the
sugar-nucleotide bond are thought to work via a double displacement
mechanism, which would require two carboxylic acid side chains (12).
Second, carboxylates could be involved in binding of substrate. Third,
the activity of ppGaNTases requires the binding of metal ions that can
adopt an octahedral geometry; ppGaNTase-T1 has a preference for
Mn2+ (13-15). Coordination of Mn2+ typically
involves multiple aspartate residues although histidine and glutamate
may also be involved (16). However, the position of key aspartate,
glutamate, and histidine residues within a glycosyltransferase model is
not known.
-sheet, flanked by
-helices. This fold most closely resembles the lac repressor protein, a member of the periplasmic binding protein family fold, which
itself is structurally related to bacteriophage T4 DNA
-glucosyltransferase. We have used site-directed mutagenesis to
mutate highly conserved and invariant aspartate, glutamate, and
histidine residues and find that those residues required for enzymatic
activity are predicted to line the active site cleft of a model based
on the lac repressor crystal structure.
EXPERIMENTAL PROCEDURES
1,4-galactosyltransferase (
4GalT) families was discovered by
PSI-BLAST (18) using the ppGaNTase family as the seed. The structural
homology to the periplasmic binding protein family was proposed based
on threading analyses with the program THREADER (19) using the human
ppGaNTase-T1 isoform as the search sequence. All runs were carried out
using default settings and scanning the full structural data base
provided with the program.
1,4-Galactosyltransferases--
The
numbers for human
4GalT-I, -II, -III, -IV, -V, and -VI are 86952, 2995442, 313298, 3132900, 2924555, and 3132904, respectively. Chicken
CK-
4GalT-I and -II are 1469908 and 1469906, respectively. Snail
4GalT is 2494837. C. elegans CE-
4GalT-A and -B are
1359573 and 4820871.
1-55 and
1-73 deletion constructs were active, the
1-73 enzyme was
labile and further deletions were not pursued. All remaining point
mutants were constructed in the
1-40 background vector.
RESULTS AND DISCUSSION
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Fig. 1.
Sequence and schematic representation of
ppGaNTase. A, sequence of the murine ppGaNTase-T1.
Solid underline indicates the transmembrane anchor,
horizontal arrow indicates the stem, broken
underlining indicates the region conserved throughout the
ppGaNTase family from worms to mammals, and the solid boxes
indicate sequence motifs that were mutated in this study. The invariant
carboxylate and histidine residues are indicated with an
asterisk. The - and
-repeats with homology to the
carbohydrate-binding sites of ricin are indicated with a dashed
box and were not mutated. N-Glycosylation sites that
were experimentally mapped (33) are circled. The soluble
form of ppGaNTase-T1 is created by proteolytic processing at residue 40 (vertical arrow). B, schematic representation of
the cytoplasmic, transmembrane, and lumenal regions of ppGaNTases. A
short N-terminal cytoplasmic tail is separated from the lumenal
catalytic domain by a transmembrane anchor (solid box) and a
stem region (cross-hatched box) which varies in length and
sequence among ppGaNTase isoforms. The catalytic unit of approximately
340 amino acids can be subdivided into two halves. The N-terminal half
of the catalytic domain is represented by a GT1 sequence motif. The
C-terminal half of the catalytic domain contains a Gal/GalNAc-T
sequence motif. The far C-terminal end of the enzyme is a region with
sequence and predicted structural homology to the plant lectin ricin
(22). Open circles indicate positions of
N-glycosylation sites.
4GalT and is therefore termed a
Gal/GalNAc-glycosyltransferase (Gal/GalNAc-T) motif. In
most but not all ppGaNTase sequence homologs, the remaining C-terminal
portion of the enzyme displays significant homology to the
-,
-,
and
-repeats of the carbohydrate-binding domains of the B chain in
the plant lectin ricin (Refs. 22 and 23, and Fig. 1, A and
B).
-strands and
-helices (Fig.
2B). The most highly conserved histidine and carboxylic acid
residues are positioned near the C-terminal ends of the
-strands. Although the primary sequence of the GT1 motif diverges among isoforms,
the JPRED program is able to produce a structural prediction based on
all known ppGaNTase family members in C. elegans (GLY3 through GLY11) and mammals (-T1 through -T5). The extreme C-terminal end of the GT1 motif in each family member contains an invariant DxH
sequence, which corresponds to the DXD sequence that has
recently been described for many other glycosyltransferases (6-9).
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Fig. 2.
Sequence and secondary structure predictions
of the GT1 sequence motif in mammalian and C. elegans
ppGaNTases. A, ClustalW alignment of the GT1
motif is shown for 14 ppGaNTases. Amino acids 119 to 227 (representing
108 of the 112 aa which comprise the GT1 motif) from the murine
ppGaNTase-T1 are indicated in the top line. C. elegans ppGaNTases are indicated as Gly proteins 3-11. Consensus
sequences are defined, using a threshold of 85% conservation and are
indicated on the consensus line and within the alignment as sequences
in reverse video print. Periods on the consensus
line indicate positions that contain a high sequence similarity but no
identity. Key aspartates, glutamates, and histidines that are invariant
are indicated by numbered positions, indicating their location in the
murine ppGaNTase protein. B, secondary structure predictions
were performed by the JPRED server. Rectangles indicate
-helices and arrows indicate
-strands. The
DXD-like sequence is a small aspect of this whole motif and
is conserved as a DXH sequence in ppGaNTases.
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Fig. 3.
Enzyme activity of GT1 motif mutants.
The murine ppGaNTase was mutated at positions described in the legend
to Fig. 2. Recombinant mutants were expressed, immunopurified, and
quantitated, as described under "Experimental Procedures." The
yield of recombinant protein was determined by SDS-PAGE analysis
(lower panel), and the enzyme activity was corrected for
recombinant protein expression. The bar chart indicates the
percent enzyme activity relative to the 1-40 wild type murine
ppGaNTase-T1 (Hatched bar). Solid bars indicate
the normalized activity of point mutants in the GT1 sequence
motif.
1,3-mannosyltransferase, and Clostridium sordellii lethal
toxin) has been mutated and found to be critical for enzyme activity
(7, 9, 11). In most GT1-containing transferases, this critical position
is part of a DXD motif. ppGaNTases, however, are exceptional
in that there is strict conservation of a DXH motif instead
(see Fig. 2A). The second conserved residue in this motif
(H211 for ppGaNTases-T1 and aspartate for most other GT1 family
members) is also critical for function. We have previously mutated H211
in bovine ppGaNTase-T1 and demonstrated that a H211A mutant is inactive
(24). A common theme among DXD motif-containing proteins is
their ability to bind nucleoside diphosphate sugars and coordinate
manganese ions (7). One possibility is that H211 is a ligand for the
metal ion. Because aspartate is the most common ligand for manganese,
we converted the ppGaNTase DXH motif into the prototypical
DXD motif by creating a murine ppGaNTase-T1 H211D mutant.
However, this mutant had no detectable activity (<0.04% relative to
wild type, Fig. 3), indicating that the aspartate could not
functionally substitute for a histidine in DXH. To test if
the H211D mutation altered the metal ion requirements of the enzyme,
the H211D mutant was assayed in the presence of 1 mM
Mg2+, Mn2+, Co 2+, and
Fe2+. No enzyme activity could be detected with these
alternative divalent metal ions (data not shown).
4GalT family of enzymes (Fig.
4, A and B). This
41-amino acid segment, the Gal/GalNAc-T motif, contains three
carboxylates in a DXXXXXWGGENXE sequence motif
(Asp-310 to Glu-322) that are invariant in the ppGaNTase family.
Mutation of these carboxylates to their amide forms (D310N, E319Q, and E322Q) reduces enzyme activity to 2, <0.04, and 1%, respectively (Fig. 4C). The WGGENXE sequence corresponds to
region II (WGGEDDD) in the
4GalT family (25, 26) and displays weaker
sequence similarity with
1,3-galactosyltransferase (
3GalT), Blood
Group B
3GalT, and the
1,3-GalNAc-transferases Blood Group A and
Forssman synthetase (9). Region II of the
4GalT family is thought to interact with both the sugar donor UDP-galactose and the sugar acceptor
(27, 28). While aspartate 310 is invariant among ppGaNTases, only the
positions containing the tryptophan, glycine, and glutamate residues
(Glu-319 and Glu-322) in the WGGENXE sequence are conserved
within the galactosyltransferase family as a WGGEDDD sequence motif.
This suggests that the critical residues Glu-319 and Glu-322 may be
involved in similar functions in both the ppGaNTase and
4GalT
families, as both families have individual isoforms capable of binding
both UDP-Gal and UDP-GalNAc (29).
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Fig. 4.
Mutagenesis of the second catalytic region of
ppGaNTases and alignments with
-1,4-galactosyltransferases. A,
ClustalW alignments of ppGaNTases from mammals and worms
(CE-GLY are C. elegans proteins). Only the
conserved sequences in the C-terminal half of the catalytic domain,
containing invariant aspartates and glutamates, are shown. Human,
murine, rat, bovine, and porcine ppGaNTase-T1 are identical in this
region. Amino acid positions 292-332, corresponding to the murine
ppGaNTase-T1, are aligned to the remainder of the ppGaNTase family. The
number symbol indicates invariant positions,
while the asterisk indicates positions that have only one
mismatch. Boxes indicate invariant carboxylic acids.
Sequences in the alignment that are either invariant or similar are
indicated by reverse video print. Similar residues are
defined in the following groupings: hydrophobic, IVALM; acidic, DE,
Basic, RHK, aromatic, FYW; polar, GNQST. B, alignment with
1,4-galactosyltransferases from humans (hum), chicken
(CK), snail and worm (CE). Reverse video
printing indicates positions that are conserved with the ppGaNTase
family. C, SDS-PAGE and activity analysis of point mutants
in the C-terminal half of the catalytic domain. Samples were processed
identically as described in the legend to Fig. 3.
-repeat, which are important for H-bonding to
hydroxyl groups of galactose in ricin (22). The phenylalanine at
position 457 aligns to an aromatic position in ricin that forms an
aromatic stacking interaction with galactose (22). However, mutation of
these positions in murine ppGaNTase-T1 did not substantially inhibit
catalytic activity of recombinant enzyme (D444H, N465A, and F457H) in
an in vitro enzyme assay (Fig. 5B). In contrast,
the analogous amino acid substitutions in ricin reduced sugar binding
by at least an order of magnitude (30). Furthermore, the double mutant
N465A/Q465A, which should affect main chain hydrogen bonding in the
ricin domain, as well as hydrogen bonding to galactose, was
characterized by only a 2-fold increase in apparent
Km for UDP-GalNAc. Collectively, these findings indicate that the C-terminal lectin domain does not participate in an
essential catalytic function as measured by in vitro
glycosylation reactions.
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Fig. 5.
Alignments and mutagenesis of the ricin-like
lectin region of murine ppGaNTase-T1. A, the -repeat
of ppGaNTase-T1 from position 430-470 is aligned to other mammalian
and nematode ppGaNTases and the sugar-binding motifs of the 2 binding
sites of ricin lectin. The most highly conserved positions in the
lectin-like region of murine ppGaNTase-T1 are indicated by
numbering and reverse video. Amino acid residues
of ricin lectin making direct contact with sugar ligand are indicated
on the lowest line and by vertical arrows.
B, activity of mutants, normalized for protein and compared
with wild type enzyme are indicated. C, SDS-PAGE of
recombinant mutants. Two weak bands are labeled in mock-treated
controls (data not shown) due to heart muscle kinase
autophosphorylation (arrows indicate heart muscle
kinase-HMK).
-Strands in a Lac Repressor Fold Topology--
A
match between the GT1 motif of ppGaNTase and the lactose repressor fold
was detected by threading analysis of some 1900 structures (score 4.21;
empirical data suggests that scores >3.5 are very significant
(THREADER Users Manual)). The lac repressor fold consists of 2 symmetrical domains, in which each domain is composed of a 5-stranded
parallel
-sheet flanked by 4
-helices (Fig.
6A) (31). The
-strands
align in a
2-
1-
3-
4-
5 order and these are flanked by 2
-helices on either side. A proposed alignment of the ppGaNTase-T1
sequence to the known three-dimensional structure of the lactose
repressor is shown in Fig. 6B. The assignment of
-strands
and
-helices agrees with the JPRED predictions depicted in Fig.
2B. The topology diagram of the lactose repressor depicts a
binding cleft formed by two symmetrical domains that face each other.
Amino acid side chains interacting with the sugar ligand are positioned
at the C-terminal ends of the strands that line the binding cleft
between the two domains (Fig. 6A). The threading of the
ppGaNTase to the lactose repressor fold demonstrates that the aa
positions essential for catalytic activity (127, 156, 209, 211, and
213) would also line the same face of the binding cleft (Fig.
6B). This mutagenesis data supports a model in which these positions form part of the sugar donor, acceptor, or
Mn2+-binding site. The crystal structure of the
bacteriophage T4 DNA
-glucosyltransferase reveals that the first
domain adopts a similar fold as the lactose repressor protein (32),
suggesting that a lactose repressor fold may be conserved among
glycosyltransferases.
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Fig. 6.
Topology diagram of lactose repressor and
hypothetical structure of the GT1 region of ppGaNTase-T1.
A, crystal structure of lactose repressor (31) indicated
that the ligand-binding site is formed by two symmetrical domains that
face each other and interlock by contributing -helices (see
-helix D and D') to each domain. Each domain is composed of a
parallel
-sheet, formed by five or six
-strands.
-Strands are
indicated as broad arrows and lie in the plane of the paper,
in a 2-1-3-4-5 order. The
-helices (shown as cylinders)
flank the
-sheet, such that the white
-helices (B and
C) are positioned behind the page and the dark
-helices
(A and D) are positioned in front of the page,
thus forming a three-layered sandwich. Residues important for binding
or interacting with substrate line the binding site at positions
located at the C-terminal ends of the
-strands
(arrowheads) or loop region just prior to the next
-helix. The N and C termini of the lactose repressor are indicated
with a N and C, respectively. The dotted
line indicates a C-terminal extension that is not as well
conserved within the periplasmic binding protein family. B,
the hypothetical structure of the N-terminal half of the ppGaNTase-T1
is predicted by structural threading. Invariant carboxylic acid and
histidine residues of the GT1 and DXD-like motifs, essential
for enzyme activity, line the predicted face of the active site.
THREADER predicts a significant match with the N-terminal half of the
catalytic domain and agrees with the assignment of
-strands and
-helices, predicted by different criteria (JPRED) in Fig. 2. The
amino acid positions at the start and end of each strand, helix, or
loop are given based on the JPRED prediction (see Fig. 2B)
and the THREADER results.
3 (data not shown). Therefore, both halves of the catalytic unit contain 3 to 4 carboxylates and 1 histidine residue that are critical for enzyme activity.
-glucosyltransferase
crystal structure indicate that substrate binding shifts the
equilibrium between open and closed forms of the protein (32).
Accordingly, mutations that interfere with the binding of one substrate
in ppGaNTases may affect binding of the other substrate through effects
on domain opening or closure. Chemical modification studies suggest
that the ppGaNTase-T1 undergoes a conformational change upon UDP-GalNAc
binding (33). Therefore, it is possible that an equilibrium between the
open and closed states is used as a mechanism to regulate ppGaNTase activity.
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ACKNOWLEDGEMENTS |
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We thank Christine Cagnina and Brian Van Wuyckhuyse for excellent technical assistance. We also thank Meng Qian for help in preparing this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DE08108 (to L. A. T.) and T35 DE07189.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 716-275-0770; Fax: 716-473-2679; E-mail: Lawrence_Tabak{at}urmc.rochester.edu.
2 F. K. Hagen, unpublished data.
3 B. Hazes and F. K. Hagen, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
ppGaNTase, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase;
4GalT,
1,4-galactosyltransferase;
3GalT,
1,3-galactosyltransferase;
GT1, glycosyltransferase motif 1;
GalT, galactosyltransferase;
PAGE, polyacrylamide gel electrophoresis;
aa, amino acid.
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REFERENCES |
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