From the Center for Oral Biology, Aab Institute for
Biomedical Sciences, University of Rochester, Rochester, New York 14642 and § INSERM Unite 377, Biologie et Physiopathologie de
Cellules Mucipares, Place de Verdun, 59045 Lille Cedex, France
Received for publication, October 23, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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We have cloned, expressed and
characterized the gene encoding a ninth member of the mammalian
UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferase (ppGaNTase) family,
termed ppGaNTase-T9. This type II membrane protein consists of a
9-amino acid N-terminal cytoplasmic region, a 20-amino acid
hydrophobic/transmembrane region, a 94-amino acid stem region, and a
480-amino acid conserved region. Northern blot analysis revealed that
the gene encoding this enzyme is expressed in a broadly distributed
manner across many adult tissues. Significant levels of 5- and
4.2-kilobase transcripts were found in rat sublingual gland, testis,
small intestine, colon, and ovary, with lesser amounts in heart, brain,
spleen, lung, stomach, cervix, and uterus. In situ
hybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene
transiently in COS7 cells resulted in no detectable transferase
activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior
action of ppGaNTase-T1), additional incorporation of GalNAc was
achieved, resulting in new hydroxyamino acid modification. The activity
of this glycopeptide transferase is distinguished from that of
ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the
MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a
hexa-glycopeptide species. This isoform thus represents the second
example of a glycopeptide transferase and is distinct from
the previously identified form in enzymatic activity as well as
expression in embryonic and adult tissues. These findings lend further
support to the existence of a hierarchical network of differential
enzymatic activity within the diversely regulated ppGaNTase family,
which may play a role in the various processes governing development.
Mucin type O-linked glycosylation is initiated by the
action of a family of UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferases (ppGaNTase,1 EC 2.4.1.41),
which catalyze the transfer of GalNAc from the nucleotide sugar
UDP-GalNAc to the hydroxyl group of either serine or threonine. A
number of functional roles for O-glycans have been suggested
(reviewed in Ref. 1), including protection from proteolytic degradation
(2), alteration of substrate structural conformation (3), aiding in
sperm-egg binding during fertilization in mice (4), and coordination of
leukocyte rolling along endothelial cells upon inflammation and injury
(5). However, the exact biological functions of O-linked
glycosylation remain largely unknown, as studies involving
chemical/enzymatic cleavage of sugars and/or mutagenesis of acceptor
residues on proteins can result in secondary effects unrelated to sugar
removal or absence. Since carbohydrates can only be "mutated"
indirectly by modifying the enzymatic activities of the
glycosyltansferases responsible for their synthesis, our efforts have
focused on the characterization of the enzyme family responsible for
the initiation of O-glycan addition.
Thus far, seven distinct mammalian isoforms from this gene family have
been identified and functionally characterized: ppGaNTase-T1 (6, 7),
-T2 (8), -T3 (9, 10), -T4 (11), -T5 (12), -T6 (13), and -T7 (14, 15).
An eighth putative isoform was ablated in mice without any obvious
phenotypic effects (16, 17); however, the enzymatic activity and the
gene encoding this isoform remain uncharacterized. Whereas some
isoforms display a broad range of expression in adult tissues and act
on a robust set of substrates (ppGaNTase-T1, -T2, and -T3), others are
more restricted in both expression and substrate preference
(ppGaNTase-T4, -T5, and -T7). ppGaNTase-T7 (14) has the distinction of
being the only transferase identified thus far that requires a
GalNAc-containing glycopeptide as a substrate; glycosylation of the
peptide substrate by ppGaNTase-T1 is required before ppGaNTase-T7 will
further glycosylate additional residues. This result indicates that not
all O-linked glycosylation occurs simultaneously and
suggests that a hierarchy of action within this family may be
responsible for the complex patterns of multisite substrate
glycosylation seen in vivo.
Here, we report the cloning of another member of this transferase
family, termed ppGaNTase-T9. In common with ppGaNTase-T7, ppGaNTase-T9
demonstrated no transferase activity against a panel of unmodified
peptide substrates in vitro. However, when the MUC5AC peptide substrate was first glycosylated by ppGaNTase-T1, the resultant
glycopeptides were readily glycosylated further by ppGaNTase-T9 in a
manner distinct from that of ppGaNTase-T7. ppGaNTase-T9 and ppGaNTase-T7 transcript expression patterns differed as well; ppGaNTase-T9 was expressed more widely across adult tissues and exhibited distinct expression patterns within developing mouse embryos.
These results suggest that glycosylation of multisite substrates occurs
through the specific and hierarchical action of multiple members of
this enzyme family, whose expression is uniquely regulated both during
development and in adult tissues.
Isolation of ppGaNTase-T9 Probes and Full-length
cDNAs--
Previously, the conserved amino acid regions EIWGGEN
and VWMDEYK were used to design sense and antisense PCR primers to
amplify products from rat sublingual gland (rat SLG) cDNA. These
products were cloned, sequenced and used to screen a rat SLG cDNA
library as described (12). A probe previously used to clone the rat ppGaNTase-T5 cDNA (12) resulted in the detection of additional isoforms when screening an oligo(dT)-primed Uni-Zap XR rat SLG cDNA
library according to standard procedures (18). A novel isoform,
designated ppGaNTase-T9, was identified by cross-hybridization with the
probe derived from positions 2076-2240 of ppGaNTase-T5 (12). One clone
containing a truncated 3' end was initially isolated (rTA-0). An
oligonucleotide (d(AGACGTTGTGGCCCAGAAAAAACTCCGAGGCTCC)) based on
the 3'-most sequence of this partial cDNA clone was end-labeled and
used to screen the cDNA library a second time. A cDNA clone containing a complete open reading frame was isolated (rTA-3). The
coding region within this clone was completely sequenced and given the
designation ppGaNTase-T9. The N-terminal transmembrane domain was
determined by a Kyte-Doolittle hydrophobicity plot.
Amino Acid Similarity Determinations--
Amino acid sequences
were aligned, one pair at a time, using the pairwise ClustalW (1.4)
algorithm in MacVector (Oxford Molecular Group). The following
alignment modes and parameters were used: slow alignment, open gap
penalty = 10, extended gap penalty = 0.1, similarity
matrix = blosum, delay divergence = 40%, and no hydrophilic
gap penalty. The percentage of amino acid sequence similarity displayed
in Tables I and II represents the sum of the percent identities and
similarities. Sequences comprising the conserved domains used in Table
I begin with the conserved region FNXXXSD in the putative
catalytic domain (amino acid position 84 in ppGaNTase-T1 (11), 100 in
ppGaNTase-T2 (8), 150 in ppGaNTase-T3 (10), 102 in ppGaNTase-T4 (11),
454 in ppGaNTase-T5 (12), 142 in ppGaNTase-T6 (13), 175 in ppGaNTase-T7
(14), and 113 in ppGaNTase-T9) and end with a conserved proline (amino acid position 425 in ppGaNTase-T1, 440 in ppGaNTase-T2, 500 in ppGaNTase-T3, 438 in ppGaNTase-T4, 796 in ppGaNTase-T5, 492 in ppGaNTase-T6, 526 in ppGaNTase-T7, and 451 in ppGaNTase-T9). The segment of conserved sequences is ~340 amino acids in length in the
various isoforms and corresponds to the putative catalytic domain based
on structural modeling and mutagenesis studies (19). Sequences aligned
in Table II consisted of the C-terminal ricin-like lectin motif (19)
(amino acids 430-558 in ppGaNTase-T1, 444-569 in ppGaNTase-T2,
505-632 in ppGaNTase-T3, 444-577 in ppGaNTase-T4, 801-929 in
ppGaNTase-T5, 498-622 in ppGaNTase-T6, 533-656 in ppGaNTase-T7, and
459-597 in ppGaNTase-T9).
Northern Blot Analysis--
Total RNA from Wistar rat tissues
was extracted according to the single-step isolation method described
by Ausubel et al. (20). Following electrophoresis in a 1%
formaldehyde-agarose gel, rat total RNA samples were transferred to
Hybond-N membranes (Amersham Pharmacia Biotech) according to Sambrook
et al. (18). A 325-bp segment of the ppGaNTase-T9 cDNA
region (from the vector pBSmTA-423, containing a 325-bp ppGaNTase-T9
insert in the HindIII site of pBluescriptKS+) from
nucleotides 1334-1756 of the amino acid coding region was labeled
using the Random Primers DNA labeling system (Life Technologies, Inc.)
according to manufacturer's instructions and used as a probe for
ppGaNTase-T9 transcripts. ppGaNTase-T7 and -T1 were detected as
described previously (12, 14). Antisense 18 S ribosomal subunit
oligonucleotide d(TATTGGAGCTGGAATTACCGCGGCTGCTGG) was end-labeled as
described (18) and used to normalize sample loading by hybridizing with
5 M excess of probe. All hybridizations were performed in
5× SSPE, 50% formamide at 42 °C with two final washes in 2× SSC,
0.1% SDS at 65 °C for 20 min.
In Situ Hybridization--
In situ hybridization
studies were performed using a modification of procedures described by
Wilkinson and Green (21). Mouse embryos were fixed overnight in freshly
prepared ice-cold 4% paraformaldehyde in phosphate-buffered saline.
The embryos were dehydrated through ethanol into xylene and embedded in
paraffin using a Tissue-Tek V.I.P. automatic processor (Miles).
Sections (5 µm) were adhered to commercially modified glass slides
(Super Frost Plus, VWR), dewaxed in xylene, rehydrated through graded
ethanols, and treated with proteinase K (to enhance probe
accessibility) and with acetic anhydride (to reduce nonspecific
background). Single-stranded RNA probes were prepared by standard
techniques with specific activities of 5 × 109
dpm/µg. ppGaNTase-T9 was detected using the plasmid pBSrT9-IS as a
template for RNA production, ppGaNTase-T7-specific RNA probes were
prepared using the plasmid pBSrT7-IS, and ppGaNTase-T1 transcripts were
detected using the plasmid pBSmT1-IS. pBSrT9-IS contains nucleotides
199-381 of the rat ppGaNTase-T9 amino acid coding region generated by
PCR amplification using the primers mTAIS+ (d(ATAGGTACCAAGCTTGCTGAACAAAGGCTGAAGGA) and mTAIS Generation of Secretion Constructs for
ppGaNTase-T9--
cDNA clones containing the 1.8-kb coding region
of ppGaNTase-T9 were isolated from the rat sublingual gland cDNA
library described previously (12). An MluI site was introduced
into cDNA clone rTA-0 by PCR amplification using the primers
rTA-MluI-S (d(CCTACGCGTCTCCTGGGGGTTCCGG)) and rTA-PCR-AS
(d(GGTCAAGCAAAGGGGGGAGCCAGTT)). This amplified product was digested
with MluI and EagI and cloned into the vector pBS-IMKF3 to create the vector, pBS-rTAmut#7. Sequencing was performed to verify that no PCR-induced mutations had been sustained in the
cloned product. A 650-bp MluI-EagI(blunt)
fragment from pBS-rTAmut#7 was then cloned into the
MluI-Bsp120(blunt) sites of pIMKF4 to generate the vector
pF4-rTA-Mut-7. (pIMKF4 is identical to pIMKF3 (11) except that the
multiple cloning site is expanded between the BglII and
NotI sites using the annealed oligonucleotides, Bgl-Not-S
(d(GATCTAGAGCTCACCGGTAAGC)) and Not-Bgl-AS (d(GGCCGCTTACCGGTGAGCTCTA)). A 1.2-kb BspEI-Bsu36I(blunt) fragment from the
cDNA clone rTA-3 was then cloned into the
BspEI-Ecl136II sites of pF4-rTA-Mut-7 to generate
the mammalian expression vector, pF4-rT9. pF4-rT9 is an SV40-based
expression vector, which generates a fusion protein containing the
following, in order: an insulin secretion signal, a metal binding site,
a heart muscle kinase site, a FLAGTM epitope tag, and the truncated
rat ppGaNTase-T9 cDNA.
Functional Expression Assays of Secreted Recombinant ppGaNTase-T9
from COS7 Cells--
COS7 cells were grown to 90% confluence in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.) + 10%
FCS at 37 °C and 5%CO2. One µg of pIMKF1 (11),
pF1-mT1 (11), pF1-rT7 (14), or pF4-rT9 and 8 µl of LipofectAMINE
(Life Technologies, Inc.) were used to transfect a 35-mm well of COS7
cells as described previously (11). Recombinant enzymes were assayed
and quantitated (data not shown) directly from the culture media of
transfected cells as described (12). The activity of ppGaNTase-T9 was
initially measured against the panel of peptide substrates described
previously (14): EA2 (PTTDSTTPAPTTK) (22), human immunodeficiency virus (RGPGRAFVTIGKIGNMR) (9), MUC2 (PTTTPISTTTMVTPTPTPTC) (23), MUC1b
(PDTRPAPGSTAPPAC) (24), EPO-T (PPDAATAAPLR) (6), rMUC-2 (SPTTSTPISSTPQPTS) (25), mG-MUC (QTSSPNTGKTSTISTT) (26), and MUC5AC
(GTTPSPVPTTSTTSAP) (27). No enzymatic activity for ppGaNTase-T9 was
detected in any of these initial assays. To generate glycopeptide substrates for analysis of ppGaNTase-T9 activity, glycosylated MUC5AC
and EA2 were prepared by incubation with Pichia
pastoris-derived recombinant ppGaNTase-T1 as described previously
(14). Briefly, Pichia-derived ppGaNTase-T1 (0.028 µg) was
incubated for 72 h at 37 °C with 1 mg of peptide in a 500-µl
total volume under the following conditions: 125 mM
cacodylate buffer (pH 7.0) containing 0.2% (v/v) Triton X-100, 12.5 mM MnCl2, 1 mM aprotinin, 1 mM leupeptin, 1 mM E64, 1 mM
phenylmethanesulfonyl fluoride, 1.25 mM AMP, 6 mM cold UDP-GalNAc. Additional enzyme (0.028 µg) and
UDP-GalNAc (3 µmol) were added at the first 24-h interval. The di-
and tri-glycosylated MUC5AC reaction products and the mono-glycosylated
EA2 reaction product were passed through AG1-X8 resin and purified on a
Waters 265 HPLC using a Vydac C-18 reverse phase column (0.46 × 25 cm) with a flow rate of 1 ml/min using a linear gradient of 5%
acetonitrile, 0.1% trifluoroacetic acid to 20% acetonitrile, 0.1%
trifluoroacetic acid in 20 min at 22 °C. Capillary electrophoresis
was performed on a model 270-HT Capillary Electrophoreses System
(Applied Biosystems, Foster City, CA) under conditions previously
described (14). MALDI-TOF mass spectra were acquired in reflector mode
(accelerating voltage: 20 kV, grid voltage: 70%) on a Voyager-DE STR
biospectrometry work station (PerSeptive Biosystems, Inc.) equipped
with delayed extraction technology. Mass spectra were externally
calibrated. Samples (1-4 pmol) were mixed with equal volume of matrix
solution (
The purified products of the reaction with Pichia-derived
ppGaNTase-T1 were used as substrates in subsequent incubations with COS7 cell-derived ppGaNTase-T9 and ppGaNTase-T7 media to generate the
data in Figs. 4-6. Equal relative amounts of each recombinant enzyme
were used in each reaction as determined by SDS-PAGE analysis (12).
Reactions were carried out in a total volume of 50 µl at the
following concentrations: 15 µg of each peptide or glycopeptide, 125 mM cacodylate buffer (pH 7.0) containing 0.2% (v/v) Triton X-100, 12.5 mM MnCl2, 1 mM
aprotinin, 1 mM leupeptin, 1 mM E64, 1 mM phenylmethanesulfonyl fluoride, and 1.25 mM
AMP. The enzyme samples were preincubated in this reaction mixture for
5 min at 37 °C, and then the reaction was initiated with the
addition of 2 nmol of UDP-[14C]GalNAc (54.7 mCi·mmol Periodate Oxidation, Sodium Borohydride Reduction, and Enzyme
Assays--
Purified MUC5AC glycopeptide (100 nmol) and MUC5AC parent
peptide (100 nmol) were oxidized with 200 µl of 0.08 M
NaIO4 in 0.05 M acetate buffer (pH 4.5) at
4 °C for 60 h, in the dark (28) in side by side reactions.
Excess periodate was destroyed by adding 20 µl of ethylene glycol.
The reaction mixtures were adjusted to pH 7.5 with 1 N
NaOH. Sodium borohydride was added to a final concentration of 0.2 M and reduction continued for 4 h at 4 °C. Excess
borohydride was destroyed by the addition of 20 µl of glacial acetic
acid, and released boric acid was evaporated several times with
methanol. The reaction mixtures were purified by HPLC as described
above. Periodate-treated and untreated MUC5AC and MUC5AC glycopeptide
were then used as substrates in reactions with COS7 cell-derived
ppGaNTase-T1, ppGaNTase-T9, or mock-transfected media (pIMKF1) as
described above to generate the data in Table III. Reactions were
performed in duplicate for 24 h at 37 °C. All reactions were
stopped by the addition of an equal volume of 10 mM EDTA. Reaction products were passed through AG1-X8 resin and eluted with 1 ml
of water, and incorporation was determined by scintillation counting.
Background values obtained from controls incubated without peptide
substrate were subtracted from each experimental value.
Radiolabeling, Digestion, and Amino Acid Sequencing of
Glycopeptide Products--
In order to determine the glycosylation
sites of products of ppGaNTase-T1, -T7, and -T9 reactions,
glycopeptides were subjected to Edman degradation on an Applied
Biosystems 473A sequencer. Samples of 2000-5000 pmol were dried on
trifluoroacetic acid washed glass fiber filters spotted with 1.5 mg of
BioBrene Plus. Amino acid phenylhydantoin (PTH)-derivatives were
chromatographed on standard ABI 5-µm C18 PTH columns using the Fast
Normal gradient program and were monitored by absorbance at 272 nm. The
PTH-Thr-O-GalNAc diastereomers were found to elute as two
peaks at unique positions in the chromatogram, near the positions of
PTH-Ser and PTH-Thr; we were unable to separate PTH-Thr from the
PTH-Thr-O-GalNAc derivative. The PTH derivative of
Ser-O-GalNAc is identified as an unresolved doublet peak
near the position of PTH-Asp (29, 30).
To confirm sites of glycosylation in the tetra-glycopeptide, the MUC5AC
tri-glycopeptide was incubated with ppGaNTase-T9 in the presence of
labeled UDP-[14C]GalNAc as described above. Products were
purified as described above and subjected to Edman degradation, where
counts were measured after each cycle.
Edman degradation sequencing of the MUC5AC hexa-glycopeptide was
confirmed by limited proteinase K digestion. Digestion of the
glycopeptide was performed in 25 µl of 0.05 M phosphate
buffer at pH 7.5 with an enzyme to substrate ratio of 1:25. The
reaction was stopped after 2 h by addition of 25 µl of 10 mM EDTA, and products were purified by HPLC as described
above and analyzed by MALDI-TOF to determine the mass of each fragment.
Fractions were then pooled according to the molecular mass and used for sequence analysis by Edman degradation.
cDNA Cloning and Sequence Analysis of ppGaNTase-T9--
A PCR
strategy based on conserved regions within the ppGaNTase family was
performed on cDNA from rat SLG; the resultant products were
purified, cloned, and sequenced to identify the nature of the insert as
described previously (12). A PCR product that previously identified the
rat ppGaNTase-T5 cDNA (12) resulted in the detection on another
novel cDNA, which shared homology to previously identified
isoforms. The cDNA clone (rTA-0) contained a 3' truncation within
the coding region. To obtain a full-length clone, an oligonucleotide
based on the 3'-most sequence of the partial cDNA clone
(d(AGACGTTGTGGCCCAGAAAAAACTCCGAGGCTCC)) was end-labeled and used to
screen the cDNA library a second time. A cDNA clone containing
a complete open reading frame was isolated (rTA-3), sequenced, and
given the designation ppGaNTase-T9.
As shown in Fig. 1, the cDNA encoding
ppGaNTase-T9 contains a 1812-bp insert encoding a unique 603-amino acid
protein. Conceptual translation of this cDNA revealed a type II
membrane protein architecture, typical of the ppGaNTase family. The
enzyme consists of a 9-amino acid N-terminal cytoplasmic region, a
20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem
region, and a 480-amino acid putative catalytic region. Table
I summarizes the degree of amino acid similarity between each of the known isoforms within the conserved putative catalytic region. ppGaNTase-T9 displays the greatest degree to
similarity within this region to ppGaNTase-T1 and the lowest degree of
similarity to ppGaNTase-hT6. Amino acid similarity within the
C-terminal ricin-like lectin motif is shown in Table II. This domain displays homology to the
carbohydrate binding region of the plant lectin, ricin, and has been
hypothesized to be involved in enzyme recognition of carbohydrate
moieties on glycopeptide substrates (19, 31). Within this region,
ppGaNTase-T9 has the greatest similarity to ppGaNTase-mT3 and the least
to ppGaNTase-mT2.
Northern Blot Analysis--
Northern blots of rat total RNA were
probed with a ppGaNTase-T9 specific probe (Fig.
2) as well as probes specific for the previously characterized ppGaNTase-T7 and -T1 (14). The highest levels
of the 5- and 4.2-kb ppGaNTase-T9 message were found in the SLG,
testis, small intestine, colon, and ovary. Smaller amounts were
detectable in heart, brain, spleen, lung, stomach, cervix, and uterus.
ppGaNTase-T7 transcripts were much more restricted in their expression,
whereas ppGaNTase-T1 transcripts were more ubiquitous, as seen
previously (14). The 18S ribosomal probe was employed to control for
RNA integrity and loading variations.
Mouse Embryonic in Situ Hybridization Analysis--
Given the
degree of amino acid conservation and nucleic acid homology for each
specific isoform across species as well as similarity of expression
patterns seen in adult tissues (14), we examined ppGaNTase-T9 gene
expression during mouse development using parasagittal sections of
embryos during late organogenesis (Theiler stage 22-23, embryonic day
14.5). The region of the rat ppGaNTase-T9 gene used as a probe is 96%
homologous to the corresponding mouse EST. Sections were hybridized
with RNA probes specific for ppGaNTase-T9, -T7 and -T1 and compared
with each other (Fig. 3). ppGaNTase-T9 is
expressed relatively abundantly compared with ppGaNTase-T7 (Fig. 3,
A versus B) and in a more restricted
pattern than ppGaNTase-T1 (Fig. 3C). A higher magnification
view of the developing hindbrain region in these animals (Fig. 3,
D-F) shows discrete accumulation of ppGaNTase-T9
transcripts in the rapidly dividing, undifferentiated ventricular zone
adjacent to the pons. Additional accumulation is observed in the
regions immediately rostral and caudal to the dorsal rhombic lips
differentiating into the cerebellum (Fig. 3D,
arrows). No accumulation is observed in the developing
choroid plexus where detectable expression of both ppGaNTase-T7 and-T1
is observed. Expression of ppGaNTase-T9 is more refined in the
hindbrain region at this stage than ppGaNTase-T1 (Fig. 3, D
versus F), with ppGaNTase-T9 transcript
accumulation colocalizing with the least differentiated tissues in this
region.
Functional Expression--
The truncated coding region of
ppGaNTase-T9 (beginning at amino acid position 39) was cloned
downstream of the insulin secretion signal, heart muscle kinase site,
and FLAGTM epitope tag in the vector pIMKF4 to generate the construct
pF4-rT9. pF4-rT9 as well as similar constructs containing a truncated
mouse ppGaNTase-T1 gene (11), a truncated rat ppGaNTase-T7 gene (14),
or no insert (pIMKF1) (11) were transfected into COS7 cells as
described previously (12). The expressed products from these
transfections were harvested from the culture media and used in
in vitro glycosylation reactions. Equivalent amounts of each
secreted product, as judged by densitometric scanning of Tricine
SDS-PAGE gels (data not shown) were used for all enzymatic assays.
Initially, no in vitro glycosylation activity was seen for
ppGaNTase-T9 (data not shown) against a panel of unmodified peptides
used previously (14). This result mirrored our initial observations
with ppGaNTase-T7, where unmodified peptides would not serve as
substrates for this enzyme. Therefore, we prepared glycosylated peptide
substrates by incubating the MUC5AC peptide with Pichia
pastoris-purified ppGaNTase-T1 enzyme for extended periods of time
in the presence of excess nucleotide sugar. The reaction products
consisting of di- and tri-substituted glycopeptides were individually
purified by HPLC and analyzed by capillary electrophoresis and mass
spectrometry to confirm their identity (Fig.
4). Purified di- and tri-glycopeptides
(m/z = 1930 and 2133, respectively) were then incubated
with equal relative amounts of recombinant ppGaNTase-T9 or ppGaNTase-T7
derived from COS7 cell culture media. The products from these reactions were then analyzed by capillary electrophoresis and mass spectrometry (Fig. 4). ppGaNTase-T9 clearly acts as a glycopeptide transferase, converting the di-glycopeptide starting material to more extensively glycosylated tri-glycopeptide (m/z = 2133) and
tetra-glycopeptide (m/z = 2337) species. ppGaNTase-T7
activity on the di-glycopeptide results in the formation of products
distinct from those formed by ppGaNTase-T9, predominantly
penta-glycopeptide (m/z = 2540) and hexa-glycopeptide
(m/z = 2744) species. When the tri-glycopeptide is used
as a substrate, ppGaNTase-T9 activity results in the formation of a
tetra-glycopeptide (m/z = 2337), even after extended
incubations, whereas ppGaNTase-T7 forms a more heavily glycosylated
hexa-glycopeptide (m/z = 2743) (Fig. 4).
The substrates and products of the aforementioned reactions were then
sequenced by Edman degradation to determine the sites of GalNAc
addition by each enzyme (Fig. 5). Fig.
5A shows the HPLC profiles for residues
1-3 and 9-13 of the MUC5AC parent peptide, the di- and
tri-glycosylated species produced by ppGaNTase-T1 and the
hexa-glycosylated species produced by ppGaNTase-T7. The * and ** denote
the diastereomeric peaks indicative of PTH-Thr-O-GalNAc, and
the *** indicates the unresolved doublet peak of
PTH-Ser-O-GalNAc. The HPLC profiles indicate that
ppGaNTase-T1 glycosylates threonines 3 and 13 in the diglycosylated
species and threonines 3, 12, and 13 in the tri-glycosylated species.
(Our earlier work indicating that T1 glycosylates serine 5 was in error
due to misinterpretation of an additional proline peak in the serine 5 HPLC profile; proline 4 in the MUC5AC sequence gave a peak near the
position of PTH-Ser-O-GalNAc, which carried over into the
serine 5 HPLC profile and was mistakenly assumed to indicate a
glycosylated serine. Since then we have repeated the Edman degradation
multiple times to conclusively assign modified positions.)
Upon incubation with the tri-glycopeptide, ppGaNTase-T7 glycosylates
threonines 2 and 10 and serine 11 to form the hexa-glycopeptide (Fig.
5A). The sites of GalNAc addition in the hexa-glycopeptide were confirmed by limited proteinase K digestion of this species and
analysis of the products by mass spectrometry and Edman degradation (Fig. 5B). The two peaks produced by this analysis
correspond to the first 9 residues of MUC5AC substituted with 2 GalNAc
residues (m/z = 1284) and the last 7 residues
substituted with 4 GalNAc residues (m/z = 1498). Edman
degradation of these fragments confirmed previous sequencing of the
unfragmented glycopeptide (data not shown). We recovered insufficient
penta-glycopeptide formed by ppGaNTase-T7 to perform sequence analysis.
The tetra-glycopeptide produced by ppGaNTase-T9 was also analyzed to
determine the site of GalNAc addition. Since the quantities of the
tetra-glycopeptide recovered were limited, we employed radiochemical
sequencing to verify the site of GalNAc addition; ppGaNTase-9 was found
to add [14C]GalNAc to threonine 2 of the tri-glycopeptide
substrate, as determined by both radioactive counting and HPLC analysis
of Edman degradation products (Fig. 5, C and D).
Unfortunately, the recovery of the tri-glycopeptide formed by
ppGaNTase-T9 was insufficient to perform sequence analysis.
To further define the requirement of the ppGaNTase-T9 isoform for a
GalNAc-containing substrate, we modified GalNAc residues by periodate
oxidation and sodium borohydride reduction. The purified glycopeptides
obtained from incubation with ppGaNTase-T1, along with the MUC5AC
parent peptide, were subjected to mild periodate oxidation followed by
sodium borohydride reduction. Periodate-treated and untreated
glycopeptides and MUC5AC parent peptide were purified by HPLC, analyzed
for integrity by capillary electrophoresis (data not shown), and
incubated with COS7 cell-derived ppGaNTase-T1, ppGaNTase-T9, or
mock-transfected (pIMKF1) media. Table
III compares the counts incorporated into
each substrate by each enzyme. The ability of ppGaNTase-T9 to use the
glycopeptide as a substrate is clearly reduced upon treatment with
periodate and sodium borohydride (compare 17080 cpm incorporated into
untreated material to 381 cpm incorporated into treated material)
(Table III). However, this reduction in incorporation by ppGaNTase-T9
is not due to the peptide being compromised during periodate treatment,
as ppGaNTase-T1 works equally well on both treated and untreated MUC5AC
(compare 21545 cpm to 18100 cpm) (Table III). These data suggest that
ppGaNTase-T9, like ppGaNTase-T7, requires the presence of intact GalNAc
on the MUC5AC peptide for it to be used as a substrate.
To begin to address the activity of these enzyme hierarchies on
peptides other than MUC5AC, we incubated P. pastoris-derived ppGaNTase-T1 with the EA2 peptide (m/z = 1340) for
extended periods of time in the presence of excess UDP-GalNAc as
described for MUC5AC. MALDI-TOF analysis and Edman degradation of the
product of this incubation indicate that ppGaNTase-T1 produces a
mono-glycopeptide (m/z = 1543) with GalNAc at threonine
7 (Fig. 6, A and
B). The same mono-glycosylated species is also produced upon
incubation of EA2 with ppGaNTase-T2 (data not shown). When this
mono-glycosylated species is then used as a substrate in subsequent
incubations with ppGaNTase-T9 or -T7, a di-glycosylated species
(m/z = 1746) is formed by both enzymes (Fig.
6A). (The additional small peak present in the ppGaNTase-T7
sample most likely represents a trace amount of tri-glycosylated
species (m/z = 1949) that was variably present and in
quantities too low to be recovered for further analysis.) Both
di-glycosylated species showed an additional GalNAc at threonine 6, indicating that ppGaNTase-T9 and -T7 are transferring GalNAc to the
same residue in this glycopeptide, producing the same final product;
this is in contrast to their respective activities on the MUC5AC
glycopeptides.
We report the cloning of a novel member of the
UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase
family, termed ppGaNTase-T9. ppGaNTase-T9 encodes a type II integral
membrane protein similar in structure to previously identified family
members. In common with ppGaNTase-T7, ppGaNTase-T9 fails to act on a
panel of unmodified peptide substrates, but rather catalyzes the
transfer of GalNAc from UDP-GalNAc to a GalNAc-containing peptide
substrate. This activity requires the prior activity of another member
of the UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferase family (ppGaNTase-T1 or
-T2) and results in the modification of an additional hydroxyamino acid
within the glycopeptide substrate. Periodate oxidation further
demonstrated that ppGaNTase-T9 also requires the presence of an intact
GalNAc residue on the glycopeptide substrate. We have determined that
ppGaNTase-T9 will act on di- and tri-glycosylated MUC5AC and
mono-glycosylated EA2, indicating that ppGaNTase-T9 will recognize
different glycoforms of a given peptide as well as more than one type
of glycopeptide substrate.
Although both ppGaNTase-T9 and ppGaNTase-T7 require glycosylated
substrates, their activities on the MUC5AC glycopeptide substrates are
clearly distinct. Whereas ppGaNTase-T7 catalyzes the formation of
hexa-glycopeptides from the tri-glycopeptide substrate, ppGaNTase-T9 produces a tetra-glycopeptide species, even after extended incubation. Edman degradation revealed that both ppGaNTase-T7 and ppGaNTase-T9 glycosylate threonine 2; however, ppGaNTase-T7 additionally acts upon
threonine 10 and serine 11. In contrast to the results with the MUC5AC
glycopeptide, ppGaNTase-T9 and -T7 act similarly on mono-glycosylated
EA2; ppGaNTase-T9 and -T7 both form a di-glycopeptide by transferring
GalNAc onto threonine 6. These results clearly demonstrate that these
two family members have both overlapping and distinct enzymatic
activities in vitro. How these in vitro observations translate in to in vivo specificities remains
to be determined.
In both the MUC5AC and EA2 glycopeptides examined, ppGaNTase-T9 adds
GalNAc to the position immediately N-terminal to a previously glycosylated residue. ppGaNTase-T7 also transfers to the position immediately N-terminal from a glycosylated threonine in the EA2 glycopeptide and N-terminal from glycosylated threonines in the MUC5AC
tri-glycopeptide. Increased glycosylation of sites vicinal to
preexisting GalNAc residues has also been observed in vitro using an undefined mixture of transferases present in human milk (32).
It is known from the analysis of mucins, that sites of glycosylation
tend to be clustered in vivo (33). This clustering may
reflect specific GalNAc recognition and subsequent local addition of
GalNAc by the glycopeptide transferases. The production of large
quantities of specifically designed glycopeptides is necessary to be
able to conclusively address the effects of number and position of
preexisting GalNAc residues on the activity and subsequent GalNAc
addition by the glycopeptide transferases.
Amino acid comparisons of all known functional mammalian ppGaNTases
have not uncovered regions of greater conservation between ppGaNTase-T7
and ppGaNTase-T9 relative to the other family members (Tables I and
II), including regions within the ricin-like lectin motif. However, a
larger panel of glycopeptide-specific enzymes on which to base
comparisons may aid in deciphering regions involved in the specific
recognition of a glycopeptide substrate. Previous work in the nematode,
Caenorhabditis elegans, identified nine ppGaNTase isoforms
(34), but enzymatic activity was detectable for only five. It is
possible that the remaining four isoforms may also require a previously
glycosylated peptide as a substrate. One recent study reports that a
single amino acid change within the ricin-like lectin motif of
ppGaNTase-T4 compromises the glycopeptide transferase activity of this
enzyme (31). However, ppGaNTase-T4 can act as both a peptide and
glycopeptide transferase, and it is unclear what specific affect this
mutation had on substrate binding and/or catalytic activity, as kinetic
parameters were not investigated.
The gene expression patterns of ppGaNTase-T9 and ppGaNTase-T7, like
their enzymatic activities, display some overlap yet are quite
distinct. By Northern analysis, ppGaNTase-T9 is broadly expressed
across many adult tissues in the rat, including the sublingual gland,
digestive tract, female reproductive tract, testis, heart, brain,
spleen, and lung. This tissue distribution is more restricted than the
near ubiquitous expression seen for ppGaNTase-T1 yet not as specific as
that seen for ppGaNTase-T7, which is found primarily in the sublingual
gland and digestive tract. Furthermore, ppGaNTase-T9 transcripts are
found in tissues where other more restricted isoforms (ppGaNTase-T5 and
ppGaNTase-T7) have not been seen (testis, lung, spleen, brain, and
heart). Within the developing embryo, expression of ppGaNTase-T9 is
quite abundant relative to ppGaNTase-T7, being found in the developing
craniofacial region, intestine, and specific regions of the hindbrain.
In contrast, ppGaNTase-T7 is only minimally expressed at this
developmental stage and is confined to very discrete regions. Earlier
embryonic stages revealed similarly disparate expression patterns for
ppGaNTase-T7 and -T9, with ppGaNTase-T9 being expressed in a
consistently broader pattern than ppGaNTase-T7 (35).
These results provide new information suggesting additional layers of
regulation in the glycosylation process. Thus far, each peptide
transferase member has displayed, to varying degrees, unique patterns
of activity in vitro (11, 12, 36, 37). The addition of a
subfamily of glycopeptide transferases displaying unique
activities may result in a potentially complex network of sequential
action and regulation within this family. The complexity of the network
is further elaborated by the spatial and temporal expression of each
transferase as well as their specific location within the Golgi
apparatus (38). For example, certain glycopeptide transferases will
only be able to act if the requisite peptide transferase has been
expressed in the same cell type at the appropriate time. Furthermore,
there is evidence suggesting that modification of preexisting GalNAc
residues by the addition of other sugars results in a decrease in the
subsequent glycosylation of other sites (32); consequently, the
respective locations within the Golgi apparatus of the glycopeptide
transferases relative to the peptide transferases and other
transferases involved in chain elongation may govern the extent to
which certain sites are glycosylated. Therefore, the specific enzymatic
activities of each isoform and their unique expression patterns in
adult tissues and during development, as well as the hierarchy of
action established within this large family, may be responsible for the
complex patterns of glycosylation observed for in vivo substrates.
One powerful approach to gain insight into the biological role of
O-linked glycosylation involves ablation of the genes
encoding these enzymes in mice. However, given the overlapping
enzymatic specificities exhibited by this family of
glycosyltransferases, single gene ablations may result in subtle or
perhaps uninformative phenotypes. There exist a number of examples
where deletions of single genes from other glycosyltransferase families
have resulted in viable, fertile mice without distinguishable
phenotypes (39). Therefore, the ablation of multiple isoforms
displaying similar enzymatic activity (e.g. ppGaNTase-T7
and-T9) and/or expression profiles may be necessary. Our current
efforts to characterize spatial and temporal expression and activity of
each member of this family will aid in making informed choices as to
which combination of gene ablations may provide insightful phenotypes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(d(ATAGAGCTCGAGAGAGCGATTCAGGGAGATT). pBSrT7-IS contains a segment of
the rat ppGaNTase-T7 (14) amino acid coding region from nucleotide
position 1759 to 1964 generated by PCR amplification using the primers
mT5IS+ (d(ATAGGTACCAAGCTTGACCAAGGGACCCGACGGATCC) and mT5IS
(d(ATAGAGCTCGAGGATGTTATTCATCTCCCACTTCTGAT). pBSmT1-IS contains
nucleotides 1376-1676 of the mouse ppGaNTase-T1 amino acid coding
region generated by PCR amplification using the primers mT1insitu+ (d(ATAGGTACCAAGCTTGTCATGGTATGGGAGGTAATCAGG)) and mT1in situ
(d(ATAGAGCTCGAGAATATTTCTGGAAGGGTGACAT)). All of the above mentioned PCR products were cloned into the KpnI and
SacI sites of pBluescript KS+. All vectors were linearized
at the introduced HindIII site and transcribed with T7 RNA
polymerase to produce labeled antisense RNA. Sections were hybridized
at Tm
15 °C, washed at high stringency
(Tm
7 °C) and treated with RNase A to further diminish nonspecific adherence of probe. Autoradiography with NBT-2 emulsion (Eastman Kodak Co.) was performed for 25 days. Slides were developed with D19 (Eastman Kodak), and the tissue counterstained with hematoxylin. Brightfield and darkfield images were
captured with a Polaroid Digital Microscope camera and processed using
Adobe Photoshop (Adobe Systems) with Image Processing Toolkit (Reindeer
Games, Asheville, NC).
-cyano-4-hydroxycinnamic acid, 10 mg/ml in 50%
acetonitrile, 0.1% trifluoroacetic acid) and deposited on sample plates.
1; 2.02 Gbq·mmol
1; 0.02 mCi·ml
1) and 20 nmol of cold UDP-GalNAc.
Reactions were performed for 96 h at 37 °C with additional
enzyme and UDP-GalNAc (22 nmol) being added after each 24-h interval.
Reactions were then stopped by the addition of an equal volume of 10 mM EDTA, purified on a Waters 265 HPLC, and analyzed by
capillary electrophoresis and MALDI-TOF as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (48K):
[in a new window]
Fig. 1.
Nucleotide and predicted amino acid sequence
of rat ppGaNTase-T9. Numbering of T9 cDNA begins with the
initiation codon. The N-terminal transmembrane domain
(underline) was determined by a Kyte-Doolittle
hydrophobicity plot. There are five putative N-glycosylation
sites, which are circled. The position of the
oligonucleotide used to introduce the MluI site in the
ppGaNTase-T9 cDNA clone is indicated above the corresponding
sequence, next to the horizontal arrow
(arrow indicates 5' to 3' orientation of the
oligonucleotide). Mismatched bases in the mutant oligonucleotide are
indicated by bold underline.
Amino acid similarity between ppGaNTase isoforms within a 340-amino
acid (aa) conserved domain
Amino acid similarity between ppGaNTase isoforms within the ricin-like
lecin motif
View larger version (59K):
[in a new window]
Fig. 2.
Northern blot analysis of ppGaNTase-T9, -T7
and -T1. Total RNA from Wistar rats was extracted from glands and
organs listed above each lane. After electrophoresis on 1%
formaldehyde-agarose gels and transfer to Hybond-N membranes, RNA was
hybridized with a ppGaNTase-T9-specific probe (T9), a
-T7-specific probe (T7), a -T1-specific probe
(T1), and an 18S rRNA probe (18S). The
T9 panel and the uppermost 18S
panel were from hybridizations of the same blot, whereas the
T7 panel, T1 panel, and lowermost
18S panel were from hybridizations of another
independent blot. Each lane contains 7.5 µg of total RNA.
Size markers are indicated on the left. SM
Gland, submandibular gland; SL Gland,
sublingual gland; Sm Intestine, small
intestine.
View larger version (151K):
[in a new window]
Fig. 3.
Expression of ppGaNTase-T9, -T7, and -T1 in
embryonic day 14.5 mouse embryos. A-C show low
magnification views of adjacent sagittal sections. D-F show
higher magnification views indicated by the boxed
region in A. A and D show
expression of ppGaNTase-T9, B and E show
expression of ppGaNTase-T7, and C and F show
expression of ppGaNTase-T1. lv, lateral ventricle;
mv, mesencephalic ventricle; mx, maxilla;
t, tongue; mn, mandible; h, heart;
i, intestine; hi, herniated intestine;
lu, lung; l, liver; p, pons;
cb, cerebellum; cp, choroid plexus;
mo, medulla oblongata. Arrows in D
represent the limits of the developing cerebellum. The bar
in A = 1 mm for A-C, and the bar
in D = 250 µm for D-F.
View larger version (21K):
[in a new window]
Fig. 4.
Capillary electrophoresis
(CE) and MALDI-TOF (MS) profiles of
MUC5AC glycopeptide substrates and reaction products. The
capillary electrophoresis and MALDI-TOF profiles of the purified
di-glycopeptide (Di) and tri-glycopeptide (Tri)
species formed by the action of Pichia-purified ppGaNTase-T1
on the MUC5AC parent peptide are shown at the top. The
profiles of the products formed by reaction with ppGaNTase-T9
(T9) or ppGaNTase-T7 (T7) are shown at the
bottom. Masses are shown next to each peak. Tri,
tri-glycopeptide; Tetra, tetra-glycopeptide;
Penta, penta-glycopeptide; Hexa,
hexa-glycopeptide.
View larger version (34K):
[in a new window]
Fig. 5.
Amino acid sequencing of MUC5AC glycopeptide
products. A, MUC5AC parent peptide (MUC5AC),
di-glycopeptide (Di) (formed by the action of ppGaNTase-T1),
tri-glycopeptide (Tri) (formed by the action of
ppGaNTase-T1), and hexa-glycopeptide (Hexa) (formed by the
action of ppGaNTase-T7) were sequenced by Edman degradation. The
residue sequenced is shown at the left, milli-absorbance
units (mAU) (at 272 nM) are shown to the
left of each profile, and retention time is shown in minutes
at the bottom. The * and ** denote the position of the
PTH-Thr-O-GalNAc diastereomers (the ** peak may represent a
combination of glycosylated and unglycosylated threonine); the ***
indicates the PTH-Ser-O-GalNAc diastereomers. T,
threonine; G, glycine; S, serine; Dm,
dimethylphenylthiourea by-product. B, MALDI-TOF mass
spectra for the fragments formed after treatment of the
hexa-glycopeptide by proteinase K. Amino acid sequences of each
fragment are shown next to each peak. Masses are shown in
parentheses. The * denotes glycosylated residues based on
Edman degradation and mass spectrometry of proteolytic fragments.
C, graph of counts incorporated at each position of the
MUC5AC tri-glycopeptide upon incubation with ppGaNTase-T9 and
[14C]UDP-GalNAc. The tetra-glycopeptide product was
subjected to Edman degradation, and counts present at each amino acid
position were determined. D, The Edman degradation profiles
for threonine 2 of the tri-glycopeptide (Tri) starting
material and tetra-glycopeptide (Tetra) product of
ppGaNTase-T9 are shown. The * and ** denote the position of the
PTH-Thr-O-GalNAc diastereomers.
ppGaNTase-T9 activity on treated and untreated MUC5AC and MUC5AC
tri-glycopeptide
View larger version (15K):
[in a new window]
Fig. 6.
MALDI-TOF profiles and Edman
degradation of EA2 and resultant EA2 glycopeptides. A,
mass spectra of EA2 mono-glycopeptide (Mono) produced by the
action of ppGaNTase-T1 and the di-glycopeptide species (Di)
produced by ppGaNTase-T9 (T9) and -T7 (T7).
Masses are shown next to each peak. Amino acid sequence of
the peptide is shown below each panel. The * denotes
glycosylated residues. B, Edman degradation profiles for
threonines 6 and 7 of the EA2 parent peptide (EA2), the
mono-glycopeptide (Mono) formed by ppGaNTase-T1, and the
di-glycopeptide (Di) formed by ppGaNTase-T7. Retention time
is shown at the bottom. The * and ** denote peaks indicative
of the PTH-Thr-O-GalNAc diastereomers. T,
threonine; Dm, dimethylphenylthiourea by-product.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DE-08108 (to L. A. T.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF241241 (rat ppGaNTase-T9).
¶ To whom correspondence should be addressed. Current address: NIDCR, National Institutes of Health, 31 Center Dr., MSC 2290, Bldg. 31, Rm. 2C39, Bethesda, MD 20892-2290. Tel.: 301-496-3571; Fax: 301-402-2185; E-mail: lawrence.tabak@nih.gov.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M009638200
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ABBREVIATIONS |
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The abbreviations used are: ppGaNTase, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase; PCR, polymerase chain reaction; SLG, sublingual gland; bp, base pair(s); kb, kilobase(s); E64, trans-epoxysuccinyl-L-leucylamido-3-methyl butane; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine; PTH, phenylthiohydantoin.
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