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INTRODUCTION |
O-Glycan is a general term for one of the carbohydrate
chains, of which synthesis is initiated by a transfer of GalNAc,
xylose, mannose, or fucose to a serine or threonine residue in
proteins. In mammalian cells, O-glycans are mostly produced
by a transfer of GalNAc through the activity of
UDP-N-acetyl-
-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase
(pp-GalNAc-T).1
O-Glycans were found in many glycoproteins, particularly in
secretory glycoproteins such as mucins. Eight core structures, core
1-8, that are basal structures initiated by the GalNAc addition to peptides, are recognized in the mucin-type O-glycan. Each
core structure is differentially expressed in conjunction with the differentiation and malignant transformation of various cells and
tissues (1-4). pp-GalNAc-Ts are biologically important because they
determine the number and position of mucin-type O-glycans in
a protein. To date, at least 11 human pp-GalNAc-Ts, -T1, -T2, -T3, -T4,
-T6, -T7, -T8, -T9, -T10, -T11, and -T12, have been identified (5-16).
The proteins are 40-60% identical in their sequence and are therefore
homologous. In particular, the predicted catalytic domains are highly
conserved. Their substrate specificities have been examined using a
variety of peptides, of which sequences are derived from native
glycoproteins. They have shown different substrate specificities,
different kinetic properties, and different tissue distributions,
although some of them showed substantial overlaps in the assessed
catalytic specificities and tissue distribution. The positions of
O-glycans in proteins are determined by a variety of
pp-GalNAc-Ts expressed in the cells and their substrate specificities. Some pp-GalNAc-Ts exhibit strong "primary activity" toward peptides that have no GalNAc, whereas others, such as pp-GalNAc-T4 and -T7,
prefer peptides having GalNAc residue(s) as acceptor substrates rather
than the corresponding peptides with no GalNAc (11, 17, 18). The
"secondary activity," i.e. GalNAc addition to a peptide already having GalNAc, of the latter may be directed by a lectin domain
in their catalytic region. It is supposed that multiple GalNAc
transfers to a single peptide are completed in the cells by a
combination of multiple pp-GalNAc-Ts having primary activity and ones
having secondary activity.
Human serum IgA consists of two structurally and functionally distinct
subclasses, IgA1 and IgA2, of which the ratio is about 85 and 15% of
total IgA, respectively (19-21). Immunoglobulin A nephropathy (IgAN),
which is the most common form of glomerulonephritis, is a serious
disease. In southern Europe, Asia, and Australia, the ratio of IgAN is
20-40% among patients with primary glomerular disease (22). Because
the method to cure IgAN is still unknown, dialysis treatment or renal
transplantation is necessary for IgAN patients in the final stage.
IgAN is characterized by the selective deposition of IgA1 in the renal
glomerular mesangium (23), although the mechanism for this is unclear.
There is some debate regarding the cause of the deposition. Several
investigators have proposed that the elevation of the serum IgA1
concentration is the cause of IgAN (24, 26). To explain why IgA1, but
neither IgA2 nor IgG is deposited, it was proposed that
O-glycan structures in the hinge region of IgA1 are
profoundly involved in the deposition. IgA1 is characterized by a long
extended polyproline structure and distinctive O-glycan side
chains in its hinge region (27, 28). The other immunoglobulins, except
for IgA1 and IgD, do not have O-glycans in their hinge
regions. Five O-glycans have been identified to attach to
the hinge region of IgA1 derived from healthy individuals (28, 29). The
amino acid positions of the O-glycans attached were
determined, and the O-glycan structure was defined to be SA
2-3Gal
1-3GalNAc
1-Ser/Thr (29). There are many reports
describing incomplete structures of O-glycans in the IgA1
hinge region of IgAN patients compared with those of normal individuals
(20, 30-35). According to such papers, the number of
O-glycans is decreased or some O-glycans are
shortened in the IgA1 hinge region of IgAN patients. Iwase et
al. (36, 37) showed by an in vitro experiment that
removal of sialic acid enzymatically resulted in the self-aggregation of IgA1, which may be the cause of the deposition of IgA1.
Based on the hypothesis that the disruption of O-glycan
synthesis in the IgA1 hinge region is the cause of IgAN, we started an
investigation of the molecular basis of O-glycan synthesis in the IgA1 hinge region. In this study, we examined, as a first step,
which pp-GalNAc-T(s) determine the initiation of O-glycan synthesis among the many members cloned to date.
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EXPERIMENTAL PROCEDURES |
Reverse Transcriptase (RT)-PCR Analysis of pp-GalNAc-T
Transcripts Expressed in Human B Cells, IgA-bearing Cells, and an IgA
Myeloma Cell Line--
Transcripts of 10 pp-GalNAc-T genes,
pp-GalNAc-T1, -T2, -T3, -T4, -T6, -T7, -T8, -T9, -T10, and
-T12, were examined by RT-PCR using an LA-PCRTM
kit (Takara, Shiga, Japan). The plasmid DNA, pCR2.1-TOPO DNA (Invitrogen) containing an individual pp-GalNAc-T gene, was
used as standard template with serial dilutions of 1, 0.1, and 0.01 pg/µl for RT-PCR. For an endogenous control, pBluescript SK(
) DNA
(Stratagene) containing the human
-actin gene was used
with a serial dilution. The primer sets for each pp-GalNAc-T
gene and the
-actin gene are listed in Table
I.
cDNAs were prepared from human lymphocytes and a cell line as
follows. Peripheral blood mononuclear cells (PBMCs) of healthy volunteers were isolated from heparinized venous blood by density gradient sedimentation over Lymphoprep (Nycomed Pharma
AS, Oslo, Norway). B cells were isolated from PBMCs using CD19 Dynabeads (Dynal,
Oslo, Norway) according to the manufacturer's protocol. To collect
IgA-positive cells, PBMCs were stained with FITC-conjugated rabbit
anti-human IgA antibody (Dako, Glostrup, Denmark), and then
FITC-positive cells were isolated using anti-FITC-conjugated magnetic
beads (Miltenyi Biotec, Bergisch Gladbach, Germany). cDNAs derived
from PBMCs, B cells (CD19+ cells), IgA-positive cells, and IgA myeloma
cells (NCI-H929) provided by American Type Culture Collection
(Manassas, VA) were synthesized using the SuperScriptTM
First-strand Synthesis System for RT-PCR (Invitrogen) according to the
instruction manual.
Construction and Purification of pp-GalNAc-T1, -T2, -T3, -T4,
-T6, and -T9 Protein Fused with FLAG Peptide--
The putative
catalytic domains of pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9 were
expressed as secreted proteins fused with a FLAG peptide in insect
cells using GATEWAYTM Cloning Technology (Invitrogen)
according to the manual supplied. PCR primers for cDNA cloning of
pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9 are shown in Table I. The
amplified fragments were inserted into pFBIF as described previously
(38). Recombinant proteins were purified with anti-FLAG M1 antibody
resin (Sigma). Expression and purification of recombinant protein were
performed as described in detail previously (14, 38).
Estimation of the Amount of FLAG-tagged Recombinant pp-GalNAc-T
Proteins--
The amount of recombinant protein recovered was
estimated by immunoblotting using the method reported in our previous
study (39). The protein was separated by SDS-PAGE (10% gel),
transferred onto polyvinylidene difluoride membranes (Immobilon,
Millipore), and probed with an anti-FLAG peptide antibody BioM2
(Sigma). The FLAG-tagged bacterial alkaline phosphatase (met-FLAG-BAP,
molecular mass of 49.2 kDa) was used as a standard to estimate the
relative amount. The pp-GalNAc-T and BAP protein bands were quantified by densitometric scanning of the digitized image using NIH image (version 1.61) software on a Macintosh computer (Apple Computer Inc.,
Cupertino, CA). The standard curve for each substrate was plotted by
increasing the amount of FLAG-tagged BAP protein on the same blotting
membrane as the pp-GalNAc-T samples. As the band intensity and the
concentration of the recombinant pp-GalNAc-T protein in the medium
exhibited a linear correlation, the amounts of recombinant pp-GalNAc-T
protein were estimated from the standard curve, using known amounts of
FLAG-tagged BAP protein (49.2 kDa). The amount of recombinant
pp-GalNAc-T protein is expressed in arbitrary units, with each unit of
intensity equivalent to 10 ng of FLAG-tagged BAP protein.
Enzyme Reaction of pp-GalNAc-T with a Peptide of the IgA1 Hinge
Region (HRP) as a Substrate--
The amino acid sequence of the human
IgA1 hinge region is reported to be VPSTPPTPSPSTPPTPSPSC (29) which
contains two mucin boxes, XTPXP (40). The
cysteine residue at the C terminus was replaced by a lysine in order to
be labeled with 5-carboxyfluorescein (5-FAM). This peptide labeled with
5-FAM was purchased from Sawady Co., Ltd. (Tokyo, Japan), and used as
an acceptor substrate.
The standard enzyme reaction mixture contained 25 mM
Tris-HCl (pH 7.4), 5 mM MnCl2, 0.1% Triton
X-100, 250 µM UDP-GalNAc (Sigma), 15 µM
acceptor substrate, and the purified enzyme in a final volume of 20 µl. The reaction mixture was incubated at 37 °C for various periods, and the reaction was terminated by boiling.
Separation of Enzyme Reaction Products by Reversed Phase-High
Performance Liquid Chromatography (HPLC)--
The reaction products
were filtered with an Ultrafree-MC column (Millipore, Bedford, MA) and
subjected to reversed phase-HPLC using a Cosmosil (5C18-AR,
4.6 × 250 mm; Nakalai Tesque, Kyoto, Japan) column. The reaction
products were eluted with solvent A (0.05% trifluoroacetic acid) and
solvent B (0.05% trifluoroacetic acid in 2-propanol/acetonitrile,
7:3). As a standard separation, the column was eluted with a linear
gradient ranging from 10 to 15% of solvent B in solvent A at a flow
rate of 1 ml/min for 40 min. With a fluorescence detector,
RF-10AXL (Shimadzu, Kyoto, Japan), FAM-labeled
glycopeptides were detected by the fluorescence intensity at 520 nm
(excitation, 492 nm).
Identification of Reaction Products by Mass Spectrometry (MS) and
Peptide Sequencing--
A method for the identification of pp-GalNAc-T
products has been reported (41-44). In order to determine the number
of GalNAc residues attached, the glycopeptides isolated by reversed
phase-HPLC were subjected to matrix-assisted laser desorption
ionization-time of flight (MALDI-TOF) MS (Reflex IV; Bruker Daltonics,
Billerica, MA) in the positive ion mode. The acceleration voltage was
20 kV, and positive ion spectra were recorded in a reflectron mode. The
concentrated glycopeptides in a 0.1% trifluoroacetic acid and matrix
solution were mixed on a stainless steel plate at 1:1. The matrix
solution contained 0.3 mg/ml of
-cyano-4-hydroxycinnamic acid in
ethanol/acetone, 2:1. The calibration of the molecular size
(m/z) was carried out using bombesin
(m/z, 1619.8), ACTH-(1-17) (m/z, 2093.1), ACTH-(18-39)
(m/z, 2465.2), and somatatostatin-28 (m/z, 3147.5). For the identification of
positions of attached GalNAc residues, Edman degradation amino acid
sequencing of glycopeptides was performed on a PPSQ-23A (Shimadzu).
With this sequencing system, a phenylthiohydantoin (PTH) derivative of
GalNAc-attached Thr (Calbiochem) was detected as two peaks eluted at
3.07 and 3.25 min, which were close to the DTT peak, and that of
GalNAc-attached Ser was identified as a peak at 2.96 min which
overlapped the PTH-Glu peak.
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RESULTS |
RT-PCR Analysis of 10 pp-GalNAc-T Transcripts in Human IgA-positive
Cells, B Cells, and IgA Myeloma Cells--
The transcripts of 10 pp-GalNAc-Ts were amplified by RT-PCR. As shown in Fig.
1, human B cells expressed six
pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, but not
pp-GalNAc-T7, -T8, -T10, or -T12. IgA-positive cells showed a similar
expression pattern, although pp-GalNAc-T9 was not detected.

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Fig. 1.
RT-PCR analysis of pp-GalNAc-T transcripts in
human IgA-positive cells, PBMC, B cells, and IgA myeloma cells.
The fluorescent intensity of PCR bands indicated the expression levels
of pp-GalNAc-Ts. Plasmid DNA containing each pp-GalNAc-T
gene was used as standard with serial dilutions of 1, 0.1, and 0.01 pg/µl. -Actin transcripts in the same cDNAs were also measured
as an internal control. The concentrations of standard plasmid for
-actin DNA were 10, 1, and 0.1 pg/µl.
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Activity Analysis of pp-GalNAc-Ts Expressed in Human B Cells by
HPLC--
By the RT-PCR analysis, six pp-GalNAc-Ts were identified as
candidates responsible for O-glycosylation of HRP in
IgA-positive cells. Therefore, the six enzymes, pp-GalNAc-T1, -T2, -T3,
-T4, -T6, and -T9, were produced as a soluble form tagged with a FLAG peptide in insect cells and purified with anti-FLAG M1 antibody agarose
gel. The amounts of pp-GalNAc-T proteins were determined by Western
blotting with anti-FLAG M2 antibody. An equal amount of each enzyme was
employed for the pp-GalNAc-T assay using a FAM-labeled IgA1 hinge
region peptide (VPSTPPTPSPSTPPTPSPSK-FAM; HRP) as an acceptor
substrate. The enzymatic reaction was performed for 15-360 min using
each enzyme, and then the reaction products of pp-GalNAc-Ts were
separated by reversed phase-HPLC (Fig.
2). The separation profiles of reaction
products differed dependent on each enzyme. In particular, the profile
of pp-GalNAc-T2 differed from those of the other five pp-GalNAc-Ts.
pp-GalNAc-T2 exhibited extremely strong activity for GalNAc transfer
compared with the other enzymes. After the incubation with pp-GalNAc-T2
for only 15 min, the acceptor substrate was completely consumed, and
multiple peaks around a retention time of 20-30 min were observed. The elution pattern of pp-GalNAc-T2 at 120 min incubation was almost the
same as that at 360 min. This indicates that the peaks at 120 min are
the final products of pp-GalNAc-T2. In the case of pp-GalNAc-T1, -T3,
-T4, -T6, and -T9, the peak of acceptor substrate, peak "S," still
remained even after 120 min of incubation, and these five pp-GalNAc-T
products were retained longer on a C18 column than the products of
pp-GalNAc-T2. Three enzymes, pp-GalNAc-T1, -T4, and -T9, showed a
similar profile but different from the profiles of pp-GalNAc-T2, -T3,
and -T6. pp-GalNAc-T1, -T4, and -T9 mainly produced a single peak of
reaction product under the conditions used, and the strength of their
O-glycosylation activity was very low. pp-GalNAc-T3 and -T6
produced several peaks that were apparently different from those of
pp-GalNAc-T2. As the activity of pp-GalNAc-T2 was extremely strong, the
peak S and the peaks close to peak S may have already shifted to
other peaks after 15 min of incubation. A more detailed experiment on
pp-GalNAc-T2 products was again performed with a shorter incubation
time (Fig. 3A). One-tenth as
much as the amount of pp-GalNAc-T2 used in the previous experiment was
employed, and the two peaks close to peak S were primarily detected
after 1 min of incubation. Eleven major peaks, numbered P1 to P11, were
observed in total during 120 min of incubation. Up to 360 min of
incubation, the amount of P11 still remained very small (data not
shown). To confirm the 11 peaks in total for pp-GalNAc-T2, we searched
for reaction conditions to obtain all these peaks in a single reaction
mixture. As seen in Fig. 3B, the reaction mixture of
pp-GalNAc-T2 gave the 11 peaks when 22.5 µM of the
acceptor substrate was used, and the incubation was terminated at 5 min
(Fig. 3B).

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Fig. 2.
Elution profiles of reaction products of each
six pp-GalNAc-T for HRP by reversed phase-HPLC. FAM-labeled IgA1
hinge peptide (VPSTPPTPSPSTPPTPSPSK-FAM; HRP) was incubated with each
enzyme for the periods indicated and separated by reversed phase-HPLC
with a C18 column. Peak S indicates the eluting position of
the acceptor substrate (HRP).
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Fig. 3.
Elution profiles obtained by reversed
phase-HPLC of the reaction products of pp-GalNAc-T2 for HRP after
incubation for the period indicated. A shows elution
profiles of reaction prod- ucts of pp-GalNAc-T2 with the indicated reaction time. The peaks
were numbered in the order of their appearance. The
1/10 and 1/2 in the
upper panels indicate the dilution ratio of enzyme.
B, pp-GalNAc-T2 produced 11 reaction products in a single
reaction under the condition. Peak S indicates the eluting
position of the acceptor substrate (non-glycosylated peptide;
HRP).
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To examine the specificity of each pp-GalNAc-T in more detail, we
improved the separation conditions with HPLC, and we focused on the
initial products of each enzyme (Fig. 4).
Fig. 4 shows initial reaction products of each enzyme early in the
incubation. pp-GalNAc-T2 produced two peaks, P1 and P2, in a minute
almost at the same time. pp-GalNAc-T1 and -T4 produced a single peak, "PX," indicated by an arrow, of which the retention time
(88.0 min) was apparently different from those of P1 and P2. This peak was commonly produced by pp-GalNAc-T1, T3, -T4, -T6, and -T9 but not by
-T2, as indicated by arrows in Fig. 4. pp-GalNAc-T3, -T6, and -T9 produced an additional peak, "PY," as indicated by
arrows in Fig. 4, at the 82.5-min retention time that
is different from that of P1 (83.0 min). These results demonstrated
that pp-GalNAc-T2 has a unique specificity toward HRP different from
that of other enzymes.

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Fig. 4.
Reversed phase-HPLC analysis of early
reaction products by pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9.
PX indicates the common peak shared by pp-GalNAc-T1, -T3,
-T4, -T6, and -T9, and PY indicates the common peak shared
by pp-GalNAc-T3, -T6, and -T9. P1 and P2 are early reaction products of
pp-GalNAc-T2.
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Acceptor Substrate Specificity of Each pp-GalNAc-T toward Various
Substrates--
As shown in Table II,
the acceptor specificity of each pp-GalNAc-T was determined with a
variety of peptides and mono-glycosylated peptides. The amount of each
recombinant pp-GalNAc-T was determined as described under
"Experimental Procedures," and the relative activity of each enzyme
for producing the first product was presented as pmol/min/unit. The
reaction was terminated with a short incubation when the first product,
of which the amount was 5-10% of the substrate, appeared. Among six
enzymes examined, pp-GalNAc-T4 is known to have a preference toward a
peptide partially glycosylated with GalNAc residues, i.e.
the secondary activity. Therefore, two mono-glycosylated peptides,
HRP-P1 and -P2, were employed to measure the secondary activity. Muc1a'
and Muc7, which are unrelated to HRP, were used as control substrates.
pp-GalNAc-T2 showed very weak activity toward Muc1a', about
one-hundredth less than the activity toward three HRP peptides. The
relative activity of pp-GalNAc-T2 for Muc1a' (0.172) was only 2.9 and
1.6 times stronger than those of pp-GalNAc-T1 (0.069) and -T3 (0.105).
In contrast, pp-GalNAc-T2 exhibited extremely strong activity not only
for non-glycosylated HRP (primary activity) but also for
mono-glycosylated HRP (secondary activity). The relative activities of
pp-GalNAc-T2 for the three HRP peptides were almost 100-500 times
stronger than those of the other enzymes. Interestingly, pp-GalNAc-T4
did not show any preference toward mono-glycosylated HRP. A significant
preference of the secondary activity of pp-GalNAc-T3 was observed for
HRP-P2 but not for HRP-P1. Muc7 was also a very good substrate for
pp-GalNAc-T2, and the other enzymes, i.e. pp-GalNAc-T1, -T3,
and -T4, also preferred Muc7 as a better substrate to Muc1a'.
Determination of the Number of GalNAc Residues Attached to HRP by
MALDI-TOF-MS--
As described above, pp-GalNAc-T2 showed the
strongest activity toward HRP and was predicted to be the most
effective pp-GalNAc-T for O-glycosylation in the IgA1 hinge
region. To identify the number of GalNAc residues attached to HRP, each
HPLC peak of the products of the pp-GalNAc-T2 was subjected to
MALDI-TOF-MS analysis.
As seen in Fig. 3B, at least 11 peaks, named P1 to P11, were
obtained. Several minor peaks were also observed, but their amounts were too small to fractionate under any of the conditions tested. Therefore, we analyzed 11 products of pp-GalNAc-T2. To recover enough
of the small peaks, such as P11, we adopted a different incubation time
or amount of enzyme for the reaction. MS spectra of all 11 peaks are
summarized in Fig. 5. [M + Na]+ ions were observed as the major ion from all
derivatives. Molecular sizes of the acceptor substrate (HRP) and GalNAc
are 2338.5 and 203.2, respectively. By the MS analysis, P1 and P2,
having a molecular mass of 2540.8, were defined as a mono-glycosylated
HRP. Thus, P3 was defined to have two GalNAc residues, and then the
results are interpreted as P4 and P5 (three GalNAcs), P6 (four
GalNAcs), P7 and P8 (five GalNAcs), P9 (six GalNAcs), P10 (seven
GalNAcs), and finally, P11 (eight GalNAcs). Although the number of
possible O-glycosylation sites in HRP is nine, the MS
analysis revealed that the maximum number of GalNAc transferred to HRP
was eight as determined in P11.

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Fig. 5.
MALDI-TOF MS analysis of 11 reaction products
of pp-GalNAc-T2. Each peak of 11 reaction products (P1
to P11) and the substrate (S) in Fig.
3B was isolated and subjected to MALDI-TOF MS analysis. S
indicates non-glycosylated acceptor substrate (HRP) and
P1-11 indicate each reaction product of pp-GalNAc-T2.
a.i., ?.
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Identification of the Positions of GalNAc Attached to HRP by
Peptide Sequencing Analysis--
Each peak of the 11 peaks in Fig.
3B was isolated and subjected to peptide sequencing to
determine the positions where GalNAc was attached in HRP (data not
shown). Based on 1) Fig. 3A which gives the order of
appearance of each peak, 2) Fig. 5 which shows the number of GalNAcs,
and 3) the results of peptide sequencing which determined the positions
of the GalNAc attached, the pathways of O-glycan initiation
by pp-GalNAc-T2 are schematically depicted in Fig.
6. In the HPLC analysis, P1 and P2
appeared at the same time at the beginning of the reaction, and the MS
analysis revealed that both peaks carried a single GalNAc residue.
Peptide sequencing revealed that the position where GalNAc was attached
in P1 was Ser-11 and that in P2 was Thr-7, as shown in Fig. 6. After
the transfer of the initial GalNAc residue at Thr-7, the second GalNAc residue was transferred to Thr-15 (P3). Three GalNAcs in P4 and P5 were
identified to be positioned at Thr-7, Thr-12, and Thr-15 (P4) and at
Thr-7, Ser-11, and Thr-15 (P5), respectively. P6, having four GalNAcs
at Thr-7, Ser-11, Thr-15, and Ser-19, was generated by the addition of
GalNAc to Ser-19 of P5. P7 was a mixture of two products, both of which
possessed five GalNAcs at Ser-3 or Thr-4, Thr-7, Ser-9, Ser-11, and
Thr-15. P8 was a product from P6 by the addition of GalNAc to Ser-9.
Thereafter, P9 and P10 were produced from P7 or P8 by the addition of
GalNAc to Ser-17. The final product of pp-GalNAc-T2, P11, was fully
glycosylated at eight positions, Ser-3, Thr-4, Thr-7, Ser-9, Ser-11,
Thr-15, Ser-17, and Ser-19.

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Fig. 6.
Schematic representation of the predicted
pathways of GalNAc transfer to HRP by pp-GalNAc-T2. The GalNAc
transfer pathways predicted from MS and peptide sequencing analysis are
shown. Amino acids with closed squares are the residues to
which the attachment of GalNAc was confirmed. Amino acids with
open squares are the residues to which GalNAc was
transferred alternatively.
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The positions of GalNAc residues of PX and PY in Fig. 4 were also
determined. PX or PY possessed a single GalNAc at Thr-12 or Thr-15,
respectively. Thus, the five enzymes showed different specificities
from pp-GalNAc-T2. pp-GalNAc-T2 did not produce a product having a
single GalNAc at Thr-12 or Thr-15 (PX or PY).
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DISCUSSION |
IgAN is defined as predominant IgA1 deposits in the renal
mesangium (45). Although there is some argument regarding the pathogenesis of IgAN, incompleteness of O-glycosylation in
the IgA1 hinge region is considered a possible cause of the deposition (20, 31, 33, 46). Unlike IgA2 or IgG, IgA1 has a structurally exceptional hinge portion that comprises a proline-rich sequence and
possesses multiple O-glycans. In the past few years, with the progress of MS analysis, capillary electrophoresis and so on, it
has been well elucidated that the number of carbohydrate residues in
the IgA1 hinge region is reduced in IgAN patients compared with normal
controls (47-49). Furthermore, it has been reported that incompletely
glycosylated IgA1 tends to aggregate in vitro (36, 50).
pp-GalNAc-Ts,
UDP-galactose:N-acetylgalactosamine-
-Ser/Thr
3-galactosyltransferase (core 1
3-Gal-T; C1Gal-T), and
2,3- and
2,6-sialyltransferases (STs) are essential for the complete synthesis of O-glycans in the IgA1 hinge region. To date, at
least 11 human pp-GalNAc-Ts (6-16), 2 human C1Gal-Ts that generate
core 1 structure, Gal
1-3GalNAc
1-Ser/Thr (51, 52), 2 human
2,3STs (53, 54) and 2
2,6STs (55, 56) have been cloned. However, it is not known which glycosyltransferases are involved in the biosynthesis of O-glycans in the IgA1 hinge region. As an
initial attempt to answer this question, we identified which
pp-GalNAc-T(s) are responsible for the biosynthesis of
O-glycans in the IgA1 hinge region. pp-GalNAc-T2 showed the
strongest pp-GalNAc-T activity toward the IgA1 hinge region among all
pp-GalNAc-Ts we examined, and transferred GalNAcs to selective Ser/Thr
residues that had been identified to be glycosylated in vivo
(29). Thus, pp-GalNAc-T2 was determined to be merely responsible for
the initiation of O-glycan synthesis in the IgA1 hinge
region in this study.
Six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were found
to be expressed in IgA-positive cells. This expression profile was
almost similar to that of the B cells and an IgA myeloma cell line. We
examined the specificity and the relative activity of the six enzymes
toward a hinge peptide (HRP) that mimicked the human IgA1 hinge region.
The retention time of HRP glycosylated by pp-GalNAc-Ts on reversed
phase-HPLC became shorter dependent on the number of GalNAcs because of
the hydrophilicity. As the number of incorporated GalNAc residues
increased, the retention time decreased. In addition, we could separate
the reaction products having the same number of GalNAcs dependent on
the positions where GalNAc was added, such as P1 versus P2,
P4 versus P5, and P7 versus P8.
As summarized in Fig. 6, two initial products, P1 and P2, were
identified to have a GalNAc at Ser-11 or Thr-7, respectively, followed
by the addition at Thr-15 as a secondary reaction. Thr-7 and Thr-15 are
typical threonine residues encoded in the mucin box,
XTPXP. Thus, the specificity of pp-GalNAc-T2 is
somehow in agreement with the mucin box rule (40), although Ser-11 is
not consistent with the rule. The existence of these two initial
products suggested that there are at least two synthetic pathways. The next product from P2 was P3; however, the product derived from P1 was
not found, and similarly some products lacked intermediates. These
transient products were thought to exist as small peaks that we could
not fractionate, and such small transient peaks may be immediately
glycosylated to the following product. P5 was considered to be
generated from both of the initial products. P4, which has a GalNAc at
Thr-12, was produced from P3; however, the products from P4 were not
detected until the end of the reaction. P4 may be a dead-end product of
the reaction.
It has also been reported that the initial
O-glycosylation of a peptide substrate influences the
subsequent glycosylation, probably due to conformational change of the
peptide and the accessibility of pp-GalNAc-Ts for particular acceptor
sites (57). There are two consecutive Ser and Thr residues, such as
Ser-3/Thr-4 and Ser-11/Thr-12, in HRP. Although one of the consecutive
residues was glycosylated, no products in which both consecutive
residues were glycosylated were found except for P11. This result
indicated that pp-GalNAc-T2 cannot glycosylate the sites next to
GalNAc-attached sites within HRP. This phenomenon is consistent
with the previous result using a peptide substrate that mimicked the
tandem repeat portion of MUC2 (41, 58). This is contrast to the
activity of pp-GalNAc-T13 which preferentially transfers GalNAc to all three consecutive Ser/Thr residues in a synthetic peptide derived from
syndecan-3 (59).
Mattu et al. (29) analyzed O-glycans in the IgA1
hinge region that was prepared from serum IgA1 of healthy individuals. They indicated that O-glycans were attached at five
positions, which correspond to Thr-4, Thr-7, Ser-9, Ser-11, and Thr-15
in our peptide, HRP. Our results indicated that pp-GalNAc-T2 is able to
transfer GalNAc residues to almost all possible glycosylation sites in
HRP. However, a tremendous amount of the enzyme and a very long
incubation were required to glycosylate all such positions. Because a
FAM-labeled short peptide was used in this study, the three-dimensional
structure might be different from the physiological structure of the
IgA1 molecule, and this may influence the enzyme specificities in an
artificial manner. The enzyme reaction was performed in
vitro; therefore, the acceptor substrate specificity of
pp-GalNAc-T2 under in vitro conditions may not necessarily reflect those of pp-GalNAc-T2 in the cells. However, the glycosylated sites of P1 to P7 except for P4 and P6 were not inconsistent with the
results obtained by analyzing natural IgA1 derived from human serum
(29). Supposing that the pathways up to P7 production reflect the
in vivo O-glycosylation, this is consistent with
the finding of Mattu et al. (29). P6 is not consistent with
the results Mattu et al. (29) because it contained GalNAc at
Ser-19; however, Ser-19 may be easily glycosylated because it exists at the end of an artificial peptide and may be difficult to be
glycosylated in the hinge region of natural IgA1 molecule.
On the other hand, all five pp-GalNAc-Ts other than pp-GalNAc-T2 showed
very weak and almost negligible activity toward HRP, and their
specificities were totally different from those of pp-GalNAc-T2. In
addition, they produced PX as an initial product that was
mono-glycosylated at Thr-12. The Thr-12 glycosylation is not consistent
with the previous report (29). These pp-GalNAc-Ts probably do not
function in physiological O-glycosylation of the IgA1 in the cells.
From the above, we concluded that pp-GalNAc-T2 is merely responsible
for the initiation of O-glycosylation in the IgA1 hinge region among the 10 pp-GalNAc-Ts that have been reported to date. A
single enzyme, pp-GalNAc-T2, can transfer GalNAc to all five positions
in the hinge region. This is quite an interesting exception from the
general rule of O-glycosylation, because it has been believed that O-glycosylation on a protein is determined by
a combination of multiple pp-GalNAc-Ts expressed in the cell. In the
case of O-glycosylation for mucins that possess a large
number of O-glycans, it is probably true that multiple
pp-GalNAc-Ts are involved in the initiation of O-glycan
synthesis on a single mucin.
The present study is the first report on the elucidation of
O-glycosylation in the IgA1 hinge region. Up-regulation or
down-regulation of the pp-GalNAc-T2 activity in the cells may lead to
the increase or decrease in the number of O-glycans in IgA1,
respectively. Based on the pathways in Fig. 6, we can predict that the
decrease of pp-GalNAc-T2 activity will result in the loss of
O-glycan in the order of Thr-4, Ser-9, Thr-15, and Thr-7 or
Ser-11. To elucidate the molecular mechanisms of incomplete
O-glycosylation of IgA1 of IgAN patients, more investigation
will be required not only for pp-GalNA-Ts but also for core 1 Gal-T(s)
and ST(s).