O-GalNAc incorporation into a cluster acceptor site of threeconsecutive threonines Distinct specificity of GalNAc-transferase isoforms Hideyuki Takeuchi1, Kentaro Kato1, Helle Hassan2, H
Trang 1O-GalNAc incorporation into a cluster acceptor site of three
consecutive threonines
Distinct specificity of GalNAc-transferase isoforms
Hideyuki Takeuchi1, Kentaro Kato1, Helle Hassan2, Henrik Clausen2and Tatsuro Irimura1
1 Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan;2Department of Oral Diagnostics, Faculty of Health Sciences, School of Dentistry, University of Copenhagen, Denmark
O-Glycosylation of three consecutive Thr residues in a
fluorescein-conjugated peptide PTTTPLK) which mimics
a portion of mucin 2) by four isozymes of
UDP-N-ace-tylgalactosaminyltransferases (pp-GalNAc-T1, T2, T3, or
T4) was investigated Partially glycosylated versions of this
peptide, PT*TTPLK, PTTT*PLK, PT*TT*PLK,
PTT*T*PLK, PT*TTPLK, and PTTT*PLK (*,
N-acetyl-galactosamine;, galactose), were also tested The products
were separated by RP-HPLC and characterized by
MALDI-TOF MS and peptide sequencing The first and the third Thr
residues act as the peptide’s initial glycosylation sites for
pp-GalNAc-T4, which were different from the sites for
pp-GalNAc-T1 and T2 (the first Thr residue) or T3 (the third
Thr residue) shown in our previous report All
pp-GalNAc-T isozymes tested exhibited distinct specificities toward
glycopeptides The most notable findings were: (a) prior incorporation of an N-acetylgalactosamine residue at the third Thr greatly enhanced N-acetylgalactosamine incor-poration into the other Thr residues when pp-GalNAc-T2, T3, or T4 were used; (b) the enhancing effect of the N-ace-tylgalactosamine residue on the third Thr was completely abrogated by galactosylation of this N-acetylgalactosamine; (c) prior incorporation of an N-acetylgalactosamine at the first Thr did not have any enhancing effect; (d) pp-GalNAc-T2 was unique as it transferred N-acetylgalactosamine into the second Thr residue only when N-acetylgalactosamine was attached to the third one
Keywords: O-glycosylation; mucin; polypeptide N-acetylga-lactosaminyltransferase; Tn antigen; UDP-GalNAc
Biosynthesis of O-glycans is mediated by the step-wise
addition of monosaccharides by a variety of
glycosyl-transferases, where topology and kinetic properties of
Golgi-resident glycosyltransferases are believed to generate
additional diversity of carbohydrate structures [1] The
initial O-glycosylation is thought to be a highly selective
process where the sequence context determines where
O-glycans are attached to proteins, although the rules
governing this selection are still poorly understood [2–11]
Mucins form a large family of membrane-associated or
secretory glycoproteins rich in O-glycans They are
pro-duced by epithelial cells and function as a physical and
biological barrier protecting mucous epithelia There are
also leukocyte and erythrocyte markers with mucin-like
structures The core polypeptides of mucins are not only
rich in serines and threonines but they also contain Ser and Thr repeats, and tandem repeats of Ser/Thr-rich stretches [12,13] Sequences with consecutive Thr and Ser residues seem to play important roles in recognition events Trun-cated O-glycans displayed on consecutive Thr residues serve
as ligands for endogenous C-type lectins on macrophages and carcinoma-specific anti-Tn antibodies [14] Many mucin-like leukocyte markers such as CD34, CD45 and CD68 bear sequences containing consecutive Ser and Thr residues at their outermost segments [15–17] Therefore, it is tempting to speculate that these consecutive Ser/Thr sequences with various arrangements of O-glycans are structural motifs having specific biological relevance [18] The first step of mucin O-glycosylation is initiated by
a family of UDP-N-acetyl-D-galactosamine : polypeptide UDP-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts,
EC 2.4.1.41) that transfer N-acetylgalactosamine(GalNAc) residues to Ser and Thr residues in a polypeptide To date, nine members of the mammalian pp-GalNAc-T family have been cloned and characterized [19–31] Although the kinetic properties and substrate specificities of some of these recombinant isozymes have been investigated by in vitro studies using several synthetic peptides as substrates, we are still far from understanding the regulation of O-glycosyla-tion [32–35] When the peptide PTTTPITTTTK [that represents a portion of the mucin 2 (MUC2) tandem repeat] was used as a substrate with detergent-soluble microsome fractions from the human colon carcinoma cell line LS174T (which expresses several members of the GalNAc-Ts family), GalNAc was transferred to these Thr
Correspondence to T Irimura, Laboratory of Cancer Biology and
Molecular Immunology, Graduate School of Pharmaceutical
Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan.
Fax: + 81 3 5841 4879, Tel.: + 81 3 5841 4870,
E-mail: irimura@mol.f.u-tokyo.ac.jp
Abbreviations: pp-GalNAc-T, UDP-N-acetyl- D -galactosaminide,
polypeptide N-acetylgalactosaminyltransferase.
Enzymes: UDP-N-acetyl- D -galactosamine : polypeptide
UDP-N-acetylgalactosaminyltransferases (EC 2.4.1.41).
(Received 19 May 2002, revised 22 August 2002,
accepted 28 October 2002)
Trang 2residues in a specific and distinct order [36,37] Also, we
reported that pp-GalNAc-T isoforms (GalNAc-T1, -T2,
and -T3) exhibited different orders of incorporation of
GalNAc residues into consecutive Thr residues of the
PTTTPLK acceptor peptide [38]
These results suggest that some pp-GalNAc-T isoforms
work in a cooperative fashion transferring to different
acceptor sites in clusters Evidence demonstrating negative
effects of GalNAc attachments for subsequent activities of
pp-GalNAc-Ts [39], suggests that the order by which
GalNAc-T isoforms initiate glycosylation may lead to
different pathways of biosynthesis resulting in different
patterns of O-glycan occupancy Furthermore, it has been
proposed recently that some GalNAc-T isoforms function
as follow-up enzymes in that they are directed by the initial
action of other isoforms [21,23,28,35,40] This latter
mech-anism is not fully understood, but recent data by Hassan
and coworkers indicate that the putative lectin domains of
these isoforms are responsible for the unique
GalNAc-glycopeptide specificities [41] We therefore hypothesized
that different subsets of pp-GalNAc-T isoforms are
desig-nated to generate different arrangement of O-glycans on
mucins having consecutive Thr residues Using a simple
model substrate with three consecutive Thr acceptor
residues, we examined whether vicinal effects, positive as
well as negative, of GalNAc and Galb1–3GalNAc residues
on the efficacy and pathway of incorporation of the second
and the third GalNAc residues with four pp-GalNAc-Ts
were observed
E X P E R I M E N T A L P R O C E D U R E S
Synthesis of acceptor substrates
A synthetic oligopeptide PTTTPLK, was used as the
acceptor substrate for the pp-GalNAc-T isozymes Its
sequence was derived from the tandem repeat domain of
the MUC2 core polypeptide (PTTTPITTTTTVTPTPTPT
GTQT) [42] It was synthesized on a Model 9020 peptide
synthesizer (Milligen, Burlington, MA, USA) with a lysine
as the C-terminal residue The peptide was labelled at
pH 7.5 (adjusted with 100 mMHepes buffer) with
fluores-cein isothiocyanate (FITC) at its N-terminal amino acid
under conditions in which the e-amino groups of lysine
residues were not modified The lysine was added to allow
further modifications to study the interaction of resultant
glycopeptides with carbohydrate recognition molecules [14]
but such experiments are not described in the present report
Using FITC–PTTTPLK as a substrate, glycopeptides
containing GalNAc residues were prepared enzymatically
Two glycopeptides, designated FITC–PT*TTPLK or
FITC–PT*TT*PLK (where * stands for a GalNAc residue),
were generated with recombinant pp-GalNAc-T1 The
remaining two glycopeptides, denoted FITC–PTTT*PLK
and FITC–PTT*T*PLK, were prepared with recombinant
pp-GalNAc-T3 Glycopeptides with Galb1–3GalNAc
resi-dues were prepared enzymatically using FITC–PT*TTPLK
or FITC–PTTT*PLK as acceptor substrates, UDP-Gal
(final 1 mM) as donor substrates, and detergent-soluble
microsome fractions of human laryngeal carcinoma H.Ep.2
cells as the source of UDP-Gal:N-acetylgalactosaminide
b1–3 galactosyltransferase(s) The incubation conditions
and the preparations of microsome fractions are described
in the following sections All glycopeptides were purified by RP-HPLC on a C18column Sites of GalNAc attachment were confirmed by protein sequencing using the PE Biosystems 490 Procise protein sequencing system [38] To test the effect of the FITC residue on the acceptor specificity
of pp-GalNAc-Ts, the same peptide without an FITC residue was synthesized, used as an acceptor substrate, and conjugated with FITC for the HPLC separation In another experiment, the same peptide with additional six alanine residues at the N terminus was synthesized, conjugated with FITC, and used as an acceptor
Preparation of recombinant pp-GalNAc-Ts Soluble recombinant pp-GalNAc-T1, T2, and T3 were prepared as described previously [43] Briefly, each of the plasmids T1-sol, pAcGP67-GalNAc-T2-sol, and pAcGP67-GalNAc-T3-sol were cotransfected with Baculo-Gold DNA (Pharmingen) to Sf9 cells The recombinant pp-GalNAc-T1, T2, and T3 were purified from the spent media pp-GalNAc-T4 was prepared from the secretions of a stably transfected Chinese hamster ovary (CHO) cell line (CHO/GalNAc-T4/21 A) as described previously [22] One unit of recombinant enzyme was defined as the amount of enzyme that transferred 1 nmol of GalNAc residues in 30 min onto FITC–PTTTPITTTTK at
a final concentration of 5 lMin 50 lL-incubation mixtures
Preparation of detergent-soluble microsome fractions
of H.Ep.2 cells Human laryngeal carcinoma H.Ep.2 cells were cultured in modified Eagle’s medium supplemented with 10% fetal bovine serum Cells were homogenized in 50 mMTris/HCl buffer pH 7.5 containing 250 mM sucrose, 1 lgÆmL)1 aprotinin (Sigma), 1 lgÆmL)1 leupeptin (Peptide Institute Inc., Osaka, Japan), and 0.5 lgÆmL)1pepstatin A (Sigma) After centrifugation at 3000 g at 4C for 10 min, the decanted supernatant was centrifuged at 100 000 g for 1 h The pellet was re-suspended in the buffer used during the homogenization containing an additional 0.1% Triton X-100 (Sigma) Protein concentrations were determined using Protein Assay Kit (Bio-Rad) with BSA as a standard The solutions were stored in aliquots at)80 C until use
Enzymatic GalNAc incorporation into peptide and glycopeptide acceptors
The standard enzyme reaction mixture consisted of 50 mM Hepes buffer pH 7.5, 5 mM MnCl2, 5 mM 2-mercapto-ethanol, 0.1% Triton X-100, 1 mM UDP-N-acetyl-D -gal-actosamine (Sigma), 5 lM acceptor peptides or glycopeptides, and 0.2 U recombinant enzyme pp-GalNAc T1, T2, T3, or T4 (0.2268 lg, 1.098 lg, 1.365 lg, and 2.768 lg, respectively), in a final volume of 100 lL Reactions were performed at 37C for 16 h and were terminated by adding 20 lL of 500 mMEDTA
Monitoring of in vitro O-glycosylation by RP-HPLC The glycosylated peptides were separated by RP-HPLC (JASCO, Tokyo, Japan) A Cosmosil column (C18,
10· 250 mm; Nacalai tesque, Japan) was used The
Trang 3column was eluted with a linear gradient ranging from 0 to
50% solvent B (0.05% trifluoroacetic acid in 70%
2-pro-panol in acetonitrile) in solvent A (0.05% trifluoroacetic
acid in water) at a flow rate of 2 mLÆmin)1 for 30 min
Eluates were monitored by fluorescence intensity at 520 nm
MALDI-TOF MS of glycosylated peptides
Glycosylated peptides were applied on a tip and mixed with
a 10 mgÆmL)1solution of a-cyano-4-hydroxycinnamic acid
dissolved in 0.1% trifluoroacetic acid/50% ethanol in water
All mass spectra were obtained on a Voyager Elite
instrument (Nippon PerSeptive Biosystems, Tokyo, Japan)
operating at an accelerating voltage of 20 kV (grid voltage
93.5%, guide wire voltage 0.05%) in the linear mode with
the delayed extraction setting Recorded data were
pro-cessed by using GRAMS/386 software
Amino acid sequencing
Pulsed liquid Edman degradation amino acid sequencing
of glycopeptides was performed with the Applied
Biosys-tems 490 Procise protein sequencing system (Perkin
Elmer) With this system, a
phenylthiohydantoin-deri-vative of GalNAc-attached Thr was identified as a pair of
peaks eluted near the positions of
phenylthiohydantoin-Ser and phenylthiohydantoin-Thr [44] Amino acid
sequencing of fully glycosylated peptide (FITC–
PT*T*T*PLK) confirmed the eluting positions The
peptides used in the present study were modified at the
N terminals and the amino acid (Pro) was not detected
The second amino acid (Thr2) was detected at the first
cycle of Edman degradation
R E S U L T S
Fractionation of products resulting from glycosylation
of FITC–PTTTPLK peptide by pp-GalNAc-T4
An FITC-labelled oligopeptide PTTTPLK that mimicked
the tandem repeat portion of MUC2 was chemically
synthesized and labelled with FITC at its N-terminal
amino acid residue Theoretically, seven different products
could be generated from this peptide upon incubation
with a pp-GalNAc-T isozyme in the presence of
UDP-N-acetyl-D-galactosamine When FITC–PTTTPLK was
incubated with recombinant pp-GalNAc-T4 for various
periods ranging up to 24 h and then subjected to
RP-HPLC, six peaks, including the unaltered peptide,
were observed depending on the incubation period
(Fig 1) These fractions (a–e) were collected separately
and analysed by MALDI-TOF MS, which showed that
the fractions corresponded to FITC–PTTTPLK bearing
either one, two, or three glycosylated residues (Fig 2)
Thus, peaks (b) and (c) apparently contained two
GalNAc residues and peaks (d) and (e) apparently
contained a single GalNAc residue Peak (a) appears to
be the fully glycosylated peptide The associated peaks on
the MALDI-TOF MS profiles are not likely to be due to
contaminating glycopeptides with smaller numbers of
attached GalNAc residues, judging from the clear
separ-ation of glycopeptides with given numbers of GalNAc
residues on the RP-HPLC These peaks in MALDI-TOF
MS profiles should be the result of degradation during the matrix-assisted ionization of these glycopeptides The degree of glycosylation depended on the duration of incubation Up to 6 h, the six peaks could be detected, with the major fraction being unglycosylated peptide At
24 h, peptides bearing one or three GalNAc residues were prominent After the addition of fresh enzyme and UDP-GalNAc, the proportion of the peptide bearing three GalNAc residues increased (data not shown)
Characterization of the pp-GalNAc-T4 glycosylation products
The sites of GalNAc attachment to the peptide were analysed by amino acid sequencing As shown in Fig 3, peak (a) isolated by RP-HPLC, indicated that all three Thr residues are glycosylated, while peak (b) contained two GalNAc residues at Thr-3 and Thr-4 Peak (d), which constituted the major peak in the HPLC, consisted of the peptide glycosylated at Thr-2 Thus, it is clear that peak (d)
is not the precursor of peak (b) Amino acid sequencing of the minor peaks corresponding to peptides with one or two GalNAc residues, namely, peaks (c) and (e), was unsuc-cessful because of their minute quantity According to their retention times, peaks (e) and (c) are likely to contain FITC– PTTT*PLK and FITC–PT*TT*PLK, respectively, although the possibility that they are FITC–PTT*TPLK and FITC–PT*T*TPLK, cannot be excluded The presence
of the three major products in this incubation mixture can
be explained by the unique acceptor specificity of pp-GalNAc-T4, which is different from the activities of pp-GalNAc-T1, T2, or T3 on FITC–PTTTPLK, as repor-ted previously [38]
Fig 1 Elution profiles of products separated by RP-HPLC after incu-bation of FITC–PTTTPLK peptide with recombinant pp-GalNAc-T4 for the indicated periods.
Trang 4Ability of partially glycosylated FITC–PTTTPLK with GalNAc to act as acceptor substrate for all four isozymes
To understand further the regulation of GalNAc transfer to consecutive Thr residues in a mucin, acceptor specificities should be investigated with a glycopeptide whose Thr residues have already been partly occupied Thus, the effects
of prior attachment of GalNAc residues to this peptide on the activities of pp-GalNAc-T1, T2, T3 or T4 were examined We enzymatically synthesized four GalNAc peptides with one or two GalNAc residues Using these four glycopeptides and FITC–PTTTPLK as acceptors (all
at a final concentration of 5 lM), GalNAc-T assays in a
100-lL reaction mixture with 0.2 U pp-GalNAc-T1, T2, T3, or T4 were performed for 16 h Incubation products were subjected to RP-HPLC (Fig 4) The separated fractions were concentrated and analysed by MALDI-TOF MS and the results are summarized in
As we had reported previously, a maximum of two, one
or three GalNAc residues was transferred onto the ungly-cosylated FITC–PTTTPLK by pp-GalNAc-T1, T2 or T3, respectively [38] When FITC–PT*TTPLK was used as an acceptor with pp-GalNAc-T1, T2, T3, or T4, 5.1%, 3.2%, 23.8%, and 10.8% of the products bore an additional GalNAc residue, respectively, while 0%, 3.4%, 3.7%, and 3.4% were fully glycosylated, respectively
When FITC–PT*TT*PLK was used as an acceptor substrate, incorporation of an additional GalNAc residue did not significantly occur with any of the pp-GalNAc-T isozymes When FITC–PTTT*PLK was used as an accep-tor, glycopeptide products with an additional GalNAc constituted 65.1%, 19.0%, 16.3%, and 10.3% of the total products for pp-GalNAc-T1, T2, T3 or T4, respectively The product resulting from the action of pp-GalNAc-T1 was FITC–PT*TT*PLK and the proportion of this product was relatively high partly because it was not converted further pp-GalNAc-T2, T3, or T4 efficiently converted FITC–PTTT*PLK into the fully glycosylated form
Fig 2 Representative profiles of MALDI-TOF MS of FITC–
PTTTPLK peptide glycosylated by recombinant pp-GalNAc-T4 and
separated by RP-HPLC Mass indicates the (M + H) + form The
profiles a–f represent the materials retrieved from peaks a–f indicated
in Fig 1 (a) The predicted mass (1755.9) corresponds to FITC–
PTTTPLK peptide with three attached GalNAc residues (band c)
The predicted mass (1552.7) corresponds to FITC–PTTTPLK peptide
with two attached GalNAc residues (d and e) The predicted mass
(1349.5) corresponds to FITC–PTTTPLK peptide with a single
attached GalNAc residue (f) The predicted mass (1146.3) corresponds
to FITC–PTTTPLK peptide with no GalNAc residue.
Fig 3 Profiles of amino acid sequencing chromatograms of FITC–
PTTTPLK peptide and its major derivatives glycosylated by
recombin-ant pp-GalNAc-T4 (A) The profile of FITC–PTTTPLK with three
GalNAc residues attached [peak (a) in Fig 1] (B) FITC–PTTTPLK
peptide with two GalNAc residues attached [peak (b) in Fig 1] (C)
FITC–PTTTPLK peptide with a single GalNAc residue attached
[peak (d) in Fig 1] (D) Untreated FITC–PTTTPLK peptide [peak (f)
in Fig 1] Asterisks indicate phenylthiohydantoin (PTH)-derivatized
a-GalNAc-Thr, which was detected as a pair of peaks.
Fig 4 Elution profiles of products separated by RP-HPLC after incu-bation of FITC–PTTTPLK peptide or its glycosylated derivatives with recombinant pp-GalNAc-T1, T2, T3, or T4 for 16 h Acceptor sub-strates were as follows: (A) FITC–PTTTPLK; (B) FITC–PT*TTPLK; (C) FITC–PT*TT*PLK; (D) FITC–PTTT*PLK; (E) FITC– PTT*T*PLK (GalNAc-Thr was indicated by T*) Broken lines indi-cate the retention time of each substrate.
Trang 5Two products containing two GalNAc residues, namely, FITC–PT*TT*PLK and FITC–PTT*T*PLK, were both generated by the action of pp-GalNAc-T2, T3, or T4 FITC–PTT*T*PLK was the apparent intermediate product
to be converted into the fully glycosylated form because FITC–PTT*T*PLK was efficiently converted to the fully glycosylated form by pp-GalNAc-T2, T3, or T4, as shown
in Table 1
Effects of galactosylation of GalNAc residues at Thr-2
or the Thr-4 Vicinal effects of attachment of a Gal residue to GalNAc at the first Thr residue (Thr-2) or the third Thr residue (Thr-4) were investigated Using FITC–PT*TTPLK and FITC– PTTT*PLK as acceptors, two glycopeptides with Galb1– 3GalNAc at Thr-2 or Thr-4 were prepared The structures
of these glycopeptides, FITC–PT*TTPLK and FITC– PTTT*PLK (T* indicates Galb1–3GalNAca-Thr), were confirmed by MALDI-TOF MS, by their binding to peanut agglutinin specific for Galb1–3GalNAc, and sensitivity to b-galactosidase from Bacillus circulans specific for 1–3 linked b-galactoside [45] Using four glycopeptides (FITC– PT*TTPLK, FITC–PT*TTPLK, FITC–PTTT*PLK and FITC–PTTT*PLK) as acceptors, GalNAc-T assays were
Table 1 Relative quantity of glycopeptides formed after incubation of
FITC-PTTTPLK with pp-GalNAc-T1, T2, T3 or T4 and UDP-GalNAc
for 16 h.
Acceptor Enzyme
Retention time (min)
Number of GalNAc attached
Percent of total products
Table 1 (Continued).
Acceptor Enzyme
Retention time (min)
Number of GalNAc attached
Percent of total products
Fig 5 Elution profiles of products separated by RP-HPLC after incu-bation of glycosylated derivatives of FITC–PTTTPLK with recombinant pp-GalNAc-T1, T2, T3, or T4 for 16 h Acceptor substrates were as follows: (A) FITC–PT*TTPLK; (B) FITC–PT*TTPLK; (C) FITC– PTTT*PLK; (D) FITC–PTTT*PLK (GalNAc-Thr and Galb1– 3GalNAc-Thr were indicated by T* and T*, respectively) Broken lines indicate the retention time of each substrate.
Trang 6performed in a 100-lL reaction mixture with 0.2 U
pp-GalNAc-T1, T2, T3, or T4 After a 16-h incubation
products were subjected to RP-HPLC (Fig 5) The peak
fractions were pooled, concentrated by evaporation, and
analysed by MALDI-TOF MS The results indicated that
the influence of prior Gal transfer on the vicinal GalNAc
transfer depended on the site and the isozyme type of
pp-GalNAc-T
1 as summarized in Table 2 Gal transfer to
GalNAc on Thr-2 did not increase the efficiency of
GalNAc-incorporation by pp-GalNAc-T1 pp-GalNAc
T2, 3, or 4, transferred GalNAc to a very low extent when
Thr-2 was occupied by GalNAc or Galb1–3GalNAc The
effect of Gal transfer to the GalNAc attached to the Thr-4
was very slight as far as pp-GalNAc-T1 is concerned
As stated in the previous sections, greater proportions of Thr-2 and Thr-3 residues in FITC–PTTT*PLK received transfer of GalNAc residues with pp-GalNAc-T2, T3, or T4 than FITC–PTTTPLK The first site of the incorporation was apparently Thr-3 then to Thr-2 This vicinal enhancing effect was abrogated by the addition of a Gal residue to the GalNAc residue in FITC–PTTT*PLK The position of the GalNAc incorporation was Thr-2 resulting in the formation
of FITC-PT*TT*PLK in the case of the action of pp-GalNAc-T1 and T2 according to the protein sequencing analysis (Fig 6)
Effects of modification of N terminals of PTTTPLK Differences in the incorporation of GalNAc into underiva-tized and the fluorescein-labeled PTTTPLK by pp-GalNAc-T2 or T3 were compared The products from the underivatized peptide were reacted with FITC and applied
to RP-HPLC The number of GalNAc incorporated residues was estimated by MALDI-TOF MS A glycopep-tide with one GalNAc residue was the predominant product after 16 h incubation with pp-GalNAc-T2 Peptide sequen-cing analysis indicated that the GalNAc residue was attached to Thr-2 Four peaks of glycopeptides with three, two, two, or one GalNAc residues were identified in the reaction mixture with pp-GalNAc-T3 By peptide sequen-cing analysis, FITC–PTT*T*PLK and FITC–PT*TT*PLK were identified as indicated in Fig 7A These results indicated that modification of the N terminus of acceptor peptides with FITC had no significant effect on the order or maximum number of attachment of GalNAc residues When FITC–PTTTPLK was used as an acceptor and incubated with pp-GalNAc-T3 for 16 h, a glycopeptide with three GalNAc residues was the major product, whereas PTTT*PLK was the major product from underivatized peptide incubated under the same conditions
Peptide AAAAAAPTTTPLK was synthesized and labelled with FITC FITC–AAAAAAPTTTPLK was
Table 2 Relative quantity of glycopeptides formed after incubation of
FITC-PTTTPLK containing a Galb1-3GalNAca residue with
pp-Gal-NAc-T1, T2, T3 or T4 and UDP-GalNAc for 16 h.
Acceptor Enzyme
Retention time (min)
Number of GalNAc attached
Percent of total products
Fig 6 Profiles of amino acid sequencing chromatograms of products after incubation of FITC–PTTT*PLK peptides with (A) recombinant pp-GalNAc-T1 or (B) pp-GalNAc-T2 and those of untreated substrates (C) FITC–PTTT*PLK and (D) FITC–PT*TTPLK Asterisks indi-cate PTH-derivatized a-GalNAc-Thr, which is detected as a pair of peaks.
Trang 7incubated with pp-GalNAc-T2 or T3 for 16 h Elution
profiles of the products on the RP-HPLC were shown in
Fig 7B Peptide sequencing analysis indicated that
pp-GalNAc-T2 transferred one GalNAc residue to Thr-8,
the first Thr in the Thr triad The first GalNAc residue
transferred b y pp-GalNAc-T3 seemed to attach to Thr-8
and Thr-10 and a product FITC–AAAAAAPT*TTPLK
did not seem to be further modified The ratio of FITC–
T*TTPLK to FITC–PT*T*T*PLK was smaller than the
ratio of FITC–AAAAAAPT*TTPLK to FITC–AAAAA
APT*T*T*PLK
D I S C U S S I O N
We hypothesize that the arrangement of O-glycans on
consecutive Ser/Thr residues in mucins and mucin-like cell
surface receptors generate structural motifs If this really is
the case, then the biosynthetic pathway of O-glycans on consecutive Ser/Thr should strictly be regulated regarding where and what order the O-glycosylation occurs In the study presented here, the initial sites of O-glycosylation and the subsequent order of attachment of GalNAc to a sequence containing three consecutive Thr residues by four glycosyltransferase isoforms were investigated The prefer-ential site of glycosylation in FITC–PTTTPLK and parti-ally modified peptides by the action of each pp-GalNAc-T are summarized in Fig 8 The initial site of GalNAc attachment to FITC–PTTTPLK with pp-GalNAc-T1, T2, and T3 was predominantly Thr-2, Thr-2, and Thr-4, respectively, as described previously [38] pp-GalNAc-T4 appears to have two preferential initial glycosylation sites, which results in the formation of FITC–PT*TTPLK [peak (d) in Fig 2] and FITC–PTTT*PLK [putative sequence
of peak (e) in Fig 2] Other investigators using various synthetic peptide acceptors have already reported that each pp-GalNAc-T has preference for different flanking amino acid sequences surrounding the Thr residue It has not been demonstrated that the order of GalNAc incorporation into three Thr and/or Ser residues in the vicinity is strictly determined Most of the previous studies focused on probability that one site was more likely to be glycosylated
Fig 7 Elution profiles of (A) PTTTPLK and (B) FITC–AAA
AAAPTTTPLK peptides (A) Elution profiles of PTTTPLK peptides
incubated with pp-GalNAc-T2 (a), pp-GalNAc-T3 (b) or buffer alone
(c), for 16 h prior to labelling with FITC on the RP-HPLC The
estimated structures of the glycopeptides corresponding to the peaks
are depicted schematically (B) Elution profiles of FITC–AAA
AAAPTTTPLK peptides incubated with pp-GalNAc-T2 (a),
pp-GalNAc-T3 (b) or buffer alone (c), for 16 h on the RP-HPLC The
estimated structures of the glycopeptides corresponding to the peaks
are depicted schematically.
Fig 8 Summary of actions of four recombinant pp-GalNAc-Ts toward TTT stretch in FITC–PTTTPLK peptide and its partially glycosylated derivatives (A) T1 (B) T2 (C) pp-GalNAc-T3, and (D) pp-GalNAc-T4 are shown The products are indicated by shaded squares according to the proportion among whole products d, Gal residues in acceptor substrates; h, GalNAc residues in acceptor substrates The percentage of GalNAc incorporated was calculated based on the total amount of acceptor substrates.
Trang 8over another site Our present results show that the order,
i.e which Thr is glycosylated first and which Thr is second,
is determined almost exclusively when a peptide sequence
and a pp-GalNAc-T are fixed The preferential pathways of
O-glycosylation of a peptide containing three consecutive
Thr residues (FITC–PTTTPLK) are indicated in Fig 9
Very interestingly and importantly, the preferential order
did not change when a 10-fold concentration of the acceptor
substrates were used and additional components were
not generated when the incubation period was extended
up to 48 h with an addition of the same amounts of
pp-GalNAc-Ts
Previous reports indicated that Pro residues positively
influenced GalNAc incorporation into a particular Thr
residue [33] Statistical studies on various peptides
contain-ing O-glycans suggested that Pro residues located at)1 and
+3 positions relative to the glycosylation site had positive
effects, although the pp-GalNAc-T having this preference
was not clear [2–5,8] The FITC–PTTTPLK used in the
present study have two Pro residues, which potentially
provide positive effects on Thr-2 according to the previous
reports [32] These Pro residues may contribute to the initial
glycosylation site by pp-GalNAc-T1 and T2 but obviously
not by T3 The specificity of each
pp-GalNAc-T seems to be unique toward consecutive pp-GalNAc-Thr residues and
their partially glycosylated derivatives For example, when
partially glycosylated FITC–PTTTPLK were used as acceptor substrates, the effect of the attached GalNAc residues on the activity of pp-GalNAc-T1 was obvious Although the initial glycosylation site for pp-GalNAc-T1 is Thr-2, this isozyme could not glycosylate Thr-2 of FITC– PTT*T*PLK Thus, the ability of pp-GalNAc-T1 to transfer GalNAc onto a Thr immediately upstream ()1)
of an existing GalNAc-Thr residue is likely to be suppressed Neither a GalNAc residue nor a Galb1–3GalNAc residue at Thr-4 of FITC–PTTTPLK significantly influenced the activity of pp-GalNAc-T1 which could transfer one GalNAc residue to Thr-2, resulting in the formation of FITC–PT*TT*PLK or FITC–PT*TT*PLK
pp-GalNAc-T2, T3, and T4 behaved differently from pp-GalNAc-T1 in that the presence of GalNAc-Thr at the penultimate position (+1) promoted their efficacy Thus, FITC–PTTT*PLK could be rapidly converted to the fully glycosylated form by all of these isozymes via the interme-diate FITC–PTT*T*PLK The preferential glycosylation of the peptide with one GalNAc residue was inhibited by the addition of a Gal residue to this GalNAc residue in FITC– PTTT*PLK
Several issues regarding the use of a relatively short peptide with fluoresceine at the N terminus as a substrate should be carefully evaluated The kinetic parameters reported for three FITC–conjugated peptides in our previ-ous publication were not distinct from those for unmodified MUC2 peptide (PTTTPISTTTMVTPTPTPTC) reported
by Wandall and coworkers [43] We also examined the specificity of detergent-soluble microsome fraction of human colon carcinoma LS174T cells towards larger GalNAc-glycosylated peptides than FITC–PTTTPLK used
in the present study [37] RT/PCR and immunocytological analysis indicated that LS174T cells expressed at least pp-GalNAc-T1, T2, T3, and T4 In vitro GalNAc-T assays were performed using FITC–PTTT*PITTTTK, FITC– PT*TTPITTTTK, FITC–PTT*T*PITT*T*TK, and FITC–PT*TTPIT*T*T*TK as substrates Similar results
on the specificity to that of our present results were also observed in these assays, although the microsome fraction contained more than two pp-GalNAc-Ts FITC– PTTT*PITTTTK were efficiently glycosylated and conver-ted to FITC–PT*T*T*PIT*T*T*T*K When FITC– PTTT*PITTTTK was used as a substrate, the order of incorporation of GalNAc residues was restricted in the formation of PT*T*T*P Within this motif, PTTT*P, a GalNAc residue was incorporated at Thr-3 at first, and after that, one more GalNAc residue was incorporated at Thr-2 Similarly, FITC–PTT*T*PITT*T*TK were converted to fully glycosylated FITC–PT*T*T*PIT*T*T*T*K Thus, the presence of extra C-terminal sequence did not seem to influence the order of GalNAc incorporation We did not examine the effect of two Pro residues on specificity of pp-GalNAc-Ts by mutation analysis, although Pro residues
in a flanking sequence may influence the initial GalNAc-attachment site in a polypeptide as mentioned above There are few previous reports regarding the acceptor specificity of GalNAc transfer by pp-GalNAc-T isozymes
on unglycosylated and partially glycosylated sequences Hanisch and coworkers reported that the addition of a GalNAc residue by pp-GalNAc-T isozymes, in particular pp-GalNAc-T2, to Ser-16 in the tandem repeat of the MUC1 mucin was accelerated when the adjacent Thr-17
Fig 9 Putative pathways of GalNAc incorporation into FITC–
PTTTPLK by the action of GalNAc-T1 (A), GalNAc-T2 (B),
pp-GalNAc-T3 (C), and pp-GalNAc-T4 (D) *, GalNAc residues; s, Gal
residues; bold arrows, reactions in which > 50% GalNAc was
incor-porated; broken arrows, the reactions in which < 50% GalNAc was
incorporated; shaded letters, hypothetical glycosylation products
which were not detected in the present investigations.
Trang 9residue was glycosylated [39,40] Bennett and coworkers
also reported that the catalytic activity of pp-GalNAc-T4
with a peptide corresponding to a MUC2 sequence was
enhanced fivefold by prior incorporation of 1–2 mole of
GalNAc by pp-GalNAc-T2 [23] However, the structural
characteristics responsible for this effect were not elucidated
In the study by Bennett and coworkers with a MUC1
peptide, pp-GalNAc-T4 preferentially transferred GalNAc
onto a Ser residue adjacent to a glycosylated Thr [21] Thus,
our findings are consistent with prior reports In addition,
we are also able now to delineate the structural basis that
regulates GalNAc incorporation into three consecutive Thr
residues
The present work indicates that GalNAc attachment to
one of three consecutive Thr residues is an important factor
that negatively or positively affects subsequent transfers of
GalNAc residues The mechanisms behind this remain to be
explored in detail, but factors should include sequence
context, influence of GalNAc residues to conformation and
recognition of acceptor and modulation of kinetic
proper-ties potentially through the lectin domain GalNAc residues
attached to the peptides via the lectin motifs contained
within their sequences, as has been postulated previously
[46] Hagen and coworkers showed that mutations in the
C-terminal ricin-like lectin motif of murine pp-GalNAc-T1
did not alter its catalytic properties [27]
Attachment of Gal to GalNAc at Thr-4 of FITC–
PTTTPLK inhibited the transfer of GalNAc to Thr-3 by
pp-GalNAc-T2, T3, and T4 This suggests that pp-Gal
NAc-T isozymes may recognize directly GalNAc residues in
the vicinity It is an interesting possibility that GalNAc-Ts
compete with glycosyltransferases responsible for the
extension of O-glycans Brockhausen and coworkers
showed that galactose incorporation by
UDP-Gal:glyco-protein-GalNAc 3-b-D-galactosyltransferase (core 1
b3-Gal-T) purified from rat liver became less efficient when
acceptor peptides were heavily converted with GalNAc
[47,48] From results to determine the glycosylation pattern
of porcine submaxillary mucin tandem repeats, Gerken and
coworkers suggested that local glycopeptide structures, such
as GalNAc density, regulate the in vivo elongation of the
O-glycan by the porcine core 1 b3-Gal-T [44,49,50]
Although many factors potentially modulate attachment
and elongation of O-glycans remain unknown, coordinated
actions of pp-GalNAc-Ts and Gal-Ts should play a major
role in generating a variety of structural motifs on
consecu-tive Thr residues The present study suggests that a decrease
in galactosylation of GalNAc residues in consecutive Thr
residues in mucins does not only expose GalNAc residues
but also promotes the formation of GalNAc clusters This
should result in an efficient binding to parasitic protozoa
such as Entamoeba histolytica through their lectins specific
for clusters of O-linked GalNAc residues [51] The O-glycan
structures of a mucin-like molecule, CD43, were shown to
be modulated upon the exposure of epithelial cells to
bac-terial lipopolysaccharides [52], which appeared to be similar
to the change observed in T cells [53] However, the present
report is one of very few to show that glycan extension
directly affects the glycosylation of backbone peptides
In conclusion, we show that a peptide mimicking a
portion of MUC2 containing three consecutive Thr residues
(FITC–PTTTPLK) can be glycosylated by pp-GalNAc-T1,
T2, T3, T4, or combinations of these isozymes, into a variety
of differently glycosylated peptides through their unique acceptor specificities Each isozyme was unique in the specificity not only to this peptide but also to the peptides with one or two GalNAc residues or Galb1–3GalNAc residues at different positions
A C K N O W L E D G E M E N T
This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (07407063, 09254101,
11557180, and 11672162), the Research Association for Biotechnology, the Program for the Promotion of Basic Research Activities for Innovative Biosciences, and the Danish Cancer Society We thank C Hiraiwa for her assistance in preparing this manuscript.
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