From the Institute of Biochemistry, University of
Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany,
the ¶ Department of Oral Diagnostics, School of Dentistry,
University of Copenhagen, DK2200 Copenhagen, Denmark, the
Macquarie Center for Analytical Biotechnology, Macquarie
University, Sydney NSW 2109, Australia, the ** Institute of Organic
Chemistry, University of Hamburg, 20146 Hamburg, Germany, and the
Institute of Medical Physics and
Biophysics, University of Münster, 48149 Münster,
Germany.
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ABSTRACT |
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In search of possible epigenetic regulatory
mechanisms ruling the initiation of O-glycosylation by
polypeptide:N-acetylgalactosaminyltransferases, we
studied the influences of mono- and disaccharide substituents of
glycopeptide substrates on the site-specific in vitro
addition of N-acetylgalactosamine (GalNAc) residues by
recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were
20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide
carrying GalNAc Although no strict sequence dependence is known for the initiation
of O-glycosylation by
polypeptide:N-acetylgalactosaminyltransferases (ppGalNAc-Ts),1 functionally
expressed recombinant enzymes display a distinct selectivity for
peptide motifs in the vicinity of putative glycosylation sites (1-3).
Until now, the prediction of O-glycosylation sites was based
on two different approaches: the analysis of in vitro or of
in vivo glycosylated peptides (4-6). Strikingly, these two
methodological strategies revealed deviating results when the patterns
of GalNAc addition to MUC1 tandem repeat peptide were analyzed (7, 8).
All five putative sites were identified as glycosylation targets
in vivo (8), whereas only Thr within the VTSA motif and/or
Thr and Ser within the GSTA motif was glycosylated in vitro
by the enzymes from tumor cells (7, 9) or milk (7) or by the
recombinant GalNAc-Ts (T1-T3) (1-3). These differences could be
explained by substrate specificities of the enzymes involved. On the
other hand, the source of ppGalNAc-Ts used in the in vitro studies and that of the in vivo processed MUC1 were the
same. However, distinct enzyme species may have been lost during
preparation or may not be active under the conditions used for in
vitro glycosylation. It could also be assumed that there is a need
for these enzymes to act spatially or temporally in specific
subcellular compartments that are not retained in the in
vitro system. Finally, a further explanation should be considered
that assumes that initial glycosylation of a peptide substrate
influences the subsequent glycosylation events at vicinal or distant
Ser/Thr positions. There are two observations that would favor this
concept: 1) although ppGalNAc-Ts have largely been localized to the
cis-Golgi (10), there are reports demonstrating a more diffuse
distribution of these enzymes throughout the Golgi system (11); 2) the
mucin core peptide can cycle between cis-Golgi and the endoplasmic
reticulum (11) and GalNAc may be added successively to incompletely
glycosylated substrates carrying short, core-type glycans. Positive or
negative influences on the acceptor qualities of the remaining, still
unglycosylated positions could be postulated to be exerted on the mono-
or oligosaccharide level and could account for the partial
unpredictability of the actual glycosylation sites. According to this
concept, the site specificity of initial O-glycosylation
would not merely be ruled by the peptide sequence around putative
target sites. Evidence for negative effects on vicinal sites induced by
mono- or disaccharide substituents has previously been reported for a
series of glycopeptide substrates based on the MUC2 repeat peptide
(12). Another in vitro glycosylation study indicated that
positive regulatory effects on vicinal sites could also be responsible
for the accelerated GalNAc transfer (7).
We followed this line of considerations by testing a panel of mono- and
disaccharide substituted peptides corresponding to one MUC1 tandem
repeat and carrying GalNAc or Gal Materials
Glycopeptides and Peptides--
The glycopeptides listed in
Table I were synthesized as described previously (13) and were analyzed
by 1H-NMR spectroscopy (400 MHz) (14) and by MALDI mass spectrometry
(15). The peptides A1 to A8 correspond to a 21-mer of the MUC1 tandem
repeat domain starting with the AHG motif and carrying one to four
O-linked disaccharides Gal Enzymes--
Polypeptide GalNAc-transferases were obtained as
follows: secreted, soluble recombinant forms of GalNAc-T1, -T2, and -T3
were expressed in insect cells as described previously (2, 3). Enzymes
were partially purified from serum-free culture supernatant of
transfected High-FiveTM (Invitrogen) cells as described
previously (3).
The ppGalNAc-transferases from human milk were enriched by
ultracentrifugation as described previously and next mixed with an
equal volume of 0.9% NaCl, 0.4% Triton X-100, containing a series of
protease inhibitors (7).
Enzymatic Assays
The peptide or glycopeptide substrates (22 nmol, 430 µM) were dried from solutions in water in a speed vac and
solubilized in 20 µl of imidazol-HCl (0.1 M), pH 7.2, containing MnCl2 (10 mM). Addition of the
cosubstrate UDP-GalNAc (300 µM) was followed by the
respective enzyme preparations (25 µl of rGalNAc-T1, 2.04 milliunits/ml; rGalNAc-T2, 4.04 milliunits/ml; rGalNAc-T3, 4.65 milliunits/ml; milk GalNAc-Ts, 1.85 milliunits/ml; specific activity measured for TAP25 as a substrate) to yield a total volume of 50 µl.
The reaction mixtures were incubated for varying time periods at
37 °C (maximum, 7 days) by adding fresh aliquots of cosubstrate and
enzyme(s) at 24-h intervals. The samples were diluted with incubation
buffer to give a total volume of 210 µl prior to ultrafiltration through ultrafree MC membranes (Millipore, Eschborn, Germany) with a
nominal cut-off of 10 kDa. Kinetic parameters were calculated by double
reciprocal Lineweaver-Burk transformations at four substrate concentrations using assay conditions described by Brockhausen et
al. (12).
Monitoring of in Vitro Glycosylation by Reversed Phase High
Performance Liquid Chromatography
Aliquots (50-100 µl) of the reaction mixtures were
injected onto a PLRP-S column (250 × 4.6 mm, Polymer
Laboratories, Shropshire, United Kingdom) or a narrow-bore ODS
Ultrasphere column (150 × 2 mm, Beckman Instruments, Munich,
Germany) and chromatographed on an HPLC system (System Gold Beckman
Instruments, Munich, Germany) by gradient elution in a mixture of
acetonitrile in water (0.1% trifluoroacetic acid) from 2% (solvent A)
to 80% (solvent B) (duration, 80 min). Alternatively, gradient elution
was performed starting from 0% solvent B to 6% solvent B (duration, 3 min), followed by a gradient from 6% solvent B to 16% solvent B
(duration, 30 min). The glycopeptides were run at 1 ml/min and detected
photometrically at 214 nm.
Measurement of GalNAc Incorporation into Glycopeptide Substrates
by Matrix-assisted Laser Desorption Ionization Mass
Spectrometry
The HPLC-purified glycopeptides were dried in a speed vac
and dissolved in a mixture of water and methanol (1:1, v/v) to yield concentration of approximately 1 mg/ml. 2,6-Dihydroxyacetophenon (concentration, 10 g/liter in 50% aqueous methanol) as a MALDI matrix
was co-crystallized with the analyte in a dried droplet preparation.
MALDI-time-of-flight experiments were performed on a VISION 2000 prototype mass spectrometer with a 2.3 m flight tube.
N2 laser at 337 nm was used. Measurements were carried out in linear mode using appropriate delay time and potential to focus the
ions of interest.
Localization of Glycosylation Sites by Edman
Degradation of Glycopeptides
Glycopeptides were sequenced on a Hewlett-Packard
G1000A protein sequencer with 3.1 (solid) chemistry that uses methanol
rather than ethyl acetate to transfer the ATZ-amino acid
(16). The Sequelon AATM membranes were from the Perseptive
Biosystems division of Perkin-Elmer. Quantitation of the
O-glycosidic serine/threonine substitution was calculated
using a glycopeptide derived from Dictyostelium discoideum
recombinant expressed glycoprotein PsA, which contains a 100%
substituted GlcNAc Structural Analyses of Terminally Glycosylated
Peptides--
The glycopeptide substrates A1-A8, A11, and A13 (Table
I) were incubated with the single or
combined recombinant enzymes (or with the GalNAc-Ts from milk) for at
least 72 h or up to 7 days in order to identify the sites and to
measure the maximal number of GalNAc residues added to the Thr/Ser
positions of the MUC1 repeat peptide. In all cases, glycopeptide
products with the maximal number of glycosylated sites were formed
within 3 days and called terminally glycosylated products. These
glycopeptides were separated from substrate or intermediate products by
reversed phase HPLC (Fig. 1) and were
analyzed by MALDI mass spectrometry (Table
II). Between one and three GalNAc
residues were transferred depending on the glycopeptide substrate
(Table II). This was calculated on the basis of pseudomolecular ions
MH+ and the mass increment of HexNAc (203.2 mass units).
Edman sequencing showed that only positions Thr-5, Ser-16, and Thr-17
could be glycosylated in vitro, and it was independent from
the glycopeptide substrate (Tables II and
III). Accordingly, the "isolated" Thr within PDTR and Ser within VTSA do not represent target sites in
vitro for the enzymes tested in the present study as was
previously shown for the unglycosylated control peptide TAP25 (7). The only exception from this general pattern was A11, which after glycosylation over a period of 7 days showed the incorporation of trace
amounts of GalNAc into the positions Ser-6/Thr-10 (pseudomolecular ion
measured at m/z 2741.5). The terminally glycosylated final products of glycopeptides A6, A7, and A8 carrying more than one disaccharide did not reveal detectable GalNAc incorporation (Table II).
The qualitative data of the site specificity of terminal glycosylation
agreed for the enzyme preparation from human milk and the combined
recombinant GalNAc-Ts (T1-T3).
Site-specific Negative Effects on Substrate Sites Mediated by
Vicinal and Distant Glycosylation--
The glycopeptides (A1-A8, A11,
and A13) and the nonglycosylated control peptide TAP25 were analyzed
for the sites of glycosylation after in vitro transfer of
the maximal number of GalNAc residues (Table III). On the basis of
these qualitative data, several site-specific effects of substrate
glycosylation on vicinal, proximal, and distant target sites were revealed.
Long Range Effects--
The terminally glycosylated product of
glycopeptide A1 did not carry GalNAc at Ser-16 (Table III). By
contrast, the nonglycosylated control peptide TAP25 and the
glycopeptide substrates, including glycopeptide A4, were glycosylated
at this position. This finding indicates that a disaccharide
substituted at Thr-5 can negatively affect a distant target site with
respect to its acceptor qualities. The long range effect, which is
exerted over a peptide stretch of 11 amino acids, is
site-specific, because a disaccharide substitution at the
adjacent Ser-6 (glycopeptide A4) had no influence on Ser-16 glycosylation (Table III). Exertion of the distance effect is also strikingly dependent on the glycan substituted at Thr-5,
i.e. glycopeptide A11 with GalNAc at Thr-5 exhibited Ser-16 glycosylation.
Vicinal Effects--
Results obtained for another group of
glycopeptide substrates demonstrated that besides long range effects
disaccharide substituents can also exert negative effects on vicinal
target sites. In glycopeptide A5 (Table I), the disaccharide at Ser-16
is located adjacent to the acceptor position Thr-17 and may exert a
negative influence, possibly mediated by steric hindrance, on the
binding of ppGalNAc-transferases and hence prevents glycosylation of
Thr-17 (Table III). A sequence variant of A5, glycopeptide A5b (Table
I), with an additional Thr in position 18 was found to be glycosylated
at this site (Table III). It can, therefore, be argued that the
negative influence of the disaccharide in Ser-16 is solely exerted
vicinal and not proximal. The relatively low amount of this minor
glycopeptide (below 20%) renders unlikely substrate competition as an
explanation for nonglycosylation of A5, the major glycopeptide in the
substrate mixture. Interestingly, a similar negative effect with
complete inhibition of vicinal glycosylation was not observed for
GalNAc addition to Ser-16 in the substrates A3 (disaccharide at Thr-17) and A4 (disaccharide at Ser-6) (Table III). It may be suggested, accordingly, that disaccharides placed on the first position of an ST
diad (here, Ser-16) could prevent the glycosylation of the second
position, whereas disaccharide on the second position of ST or TS diads
(here, Thr-17 or Ser-6) may not necessarily inhibit addition to the first.
Proximal Effects--
Ser-16 glycosylation was not affected by
disaccharide substitution at the proximal position Effects Induced by Multiple Glycosylation--
Remarkably,
substrate peptides substituted with more than one disaccharide (A6, A7,
and A8) could not serve as acceptors for rGalNAc-Ts or milk GalNAc-Ts
at any of the remaining possible positions (Table III). With respect to
positions Ser-6 and Thr-10, which are not in vitro target
sites for rGalNAc-T1 to -T3 or milk GalNAc-Ts, this finding excludes
the possible existence of positive regulatory mechanisms mediated by
core1-disaccharides. On the other hand, the negative effect on Ser-16
glycosylation in A6 induced by disaccharide substitution at Thr-5 and
Thr-17 is solely exerted on the disaccharide level, because the
structural analogue of the glycopeptide, the monosaccharide substituted
glycopeptide A11, was a substrate of ppGalNAc-Ts (Table III). Most
likely, the prevention of Ser-16 glycosylation in A6 is caused by
disaccharide substitution of Thr-5 and is exerted in the same way as
the negative long range effect on Ser-16 glycosylation in glycopeptide
A1. It should be recalled at this point that vicinal substitution with
disaccharide at Thr-17 did not suppress Ser-16 glycosylation in A3
(Table III).
Kinetic Studies--
The rates of GalNAc incorporation into the
three actual in vitro glycosylation sites of MUC1 repeat
peptide (Thr-5, Ser-16, and Thr-17) were different for each position
and ppGalNAc-T used. Kinetic studies were performed by quantitative
HPLC/Edman sequencing and by radiometric measurement of
[14C]GalNAc incorporation to determine the apparent
kinetic constants. This approach allowed the identification and
quantitation of each intermediate or final glycopeptide product. It was
also possible to assign for some of the glycopeptide substrates
apparent Km and Vmax values
to distinct glycosylated sites.
Site Preferences of ppGalNAc-Ts--
The ppGalNAc-Ts from milk
glycosylated Thr-5 at higher rates than Thr-17 or Ser-16. This is
evidenced from previous HPLC/Edman analysis of TAP25 products (7), but
also from the apparent kinetic constants (Table III) obtained for
glycopeptide substrates A1 (glycosylation of Thr-17; see also Fig.
2C), A3 (mainly glycosylation of Thr-5; see also Fig. 2,
A and C), and A5 (glycosylation of Thr-5). The
lowest relative reaction rates were measured for the Ser-16 position,
which made glycosylation of this site the rate-limiting step in the
glycosylation of peptides A2, A3, A4, A13, and TAP25 (Fig. 2,
A and B). Accordingly, no Ser-16 glycosylation
was generally found in HPLC fractions corresponding to intermediate
products. The same relative rates of GalNAc incorporation into the
three positions were found when using the recombinant enzymes in a
mixture. The single rGalNAc-T species exhibited distinct preferences
for the in vitro glycosylation
sites within the VTSA and GSTA motifs, respectively (Tables IV and
V). As demonstrated previously for rGalNAc-T1 and -T3 (1), the best substrates for these enzymes are
characterized by a nonglycosylated Thr-5 (compare A3 to A1 in Table
IV), whereas rGalNAc-T2 prefers the nonglycosylated GSTA motif (compare
A1 to A3 and A5 in Table IV). rGalNAc-T2 is the enzyme species that
adds GalNAc most efficiently to Ser-16 (glycosylation of A11). This is
also evident from HPLC analysis of the final product of A3
glycosylation (Table V). Whereas the formation of A3(Thr-5,
Ser-16)-GalNAc2 catalyzed by the rGalNAc-T1 and rGalNAc-T3 did not exceed 7% of the total glycopeptides (substrate and products) after reaction times of 72 h, the same product represented 28% of
total glycopeptide using rGalNAc-T2 (Table V).
Negative Glycosylation-induced Effects--
Glycosylation of
Ser-16 is apparently the most sensitive indicator of effects induced by
a series of glycosylation events at the proximal sites Positive Glycosylation-induced Effects--
Besides negative
effects exerted by glycan substituents on vicinal, proximal and distant
sites, the acceleration of reaction rates was also demonstrated to be
induced by vicinal glycosylation. Position Ser-16, which except for
rGalNAc-T2 is reportedly a poor substrate of the GalNAc-Ts (3), becomes
a significantly better target site after substitution of the vicinal
Thr-17 with GalNAc (glycopeptide A11, Fig. 2A). Accordingly,
for TAP25 lacking a GalNAc substitution at Thr-17 the percentage of
Ser-16 glycosylated products measurable after 24 h was below 2%.
When the glycopeptide A11 was used as a substrate, around 17% of
Ser-16 positions were glycosylated during the same time (Fig.
2A). Whereas with TAP25 a maximal incorporation of 15% was
reached after 72 h, about 42% of Ser-16 glycosylated products
were detected with A11. The positive effect described above is exerted
only on the monosaccharide level (see Ser-16 glycosylation of A3 in
Fig. 2A).
Previous in vitro studies of initial
O-glycosylation of MUC1 repeat peptides revealed that only
three out of five putative sites were targets for the ppGalNAc-Ts from
different sources and recombinant forms of the first three cloned
rGalNAc-Ts (1-3, 7, 9). This in vitro pattern, however,
contrasted strikingly with the glycosylated sites identified on VNTR
glycopeptides derived from purified milk MUC1, which exhibited at least
partial glycosylation of five potential glycosylation positions (8).
These observations prompted us to look for possible explanations that
could abrogate this discrepancy: 1) undefined site-specific ppGalNAc-Ts
may exist that could add the sugar residue to the positions Ser-6
within the VTSA motif or to Thr-10 within the DTRP motif, 2) the assay conditions might be not optimal for the respective enzymes used, or 3)
glycosylation at one site could influence the substrate qualities at
other potential O-glycosylation sites. The latter influence
could be expected to be negative due to a glycosylation-induced decrease in the accessibility of the peptide to the enzymes. On the
other hand, only positive glycosylation-induced effects on poor
substrate positions would explain the higher degree of site occupation
found in vivo.
The reported results clearly demonstrate that all of the above
assumptions can be regarded as valid in the in vitro model of MUC1 glycosylation. Negative glycosylation effects on the GalNAc transfer into vicinal sites, but also into distant sites, were observed
on the disaccharide level of glycopeptide substrates (see Table III).
The stimulation exerted by GalNAc substitution of Thr-17 in GSTA on the
vicinal Ser glycosylation was the only positive effect detected in this
model (see Fig. 2, A and B). Accordingly, poor
target sites Ser-6 and Thr-10 remained unglycosylated on all peptide
and glycopeptide substrates, even when reaction time was extended to 7 days. Traces of GalNAc added to one of these sites by milk enzymes
after such a long reaction time may indicate that the kinetics of the
stochastic in vitro glycosylation deviates by orders of
magnitude from that of in vivo, which occurs at a highly
organized membraneous surface. These considerations may explain the
lack of in vitro glycosylation of Ser-6 and Thr-10. On the
other hand, results with novel rGalNAc-Ts show that among these
enzymes, rGalNAc-T4 (17) exhibits specificity for these two sites
preferentially, with peptide substrates substituted already with GalNAc
at Thr-5, Ser-16 and Thr-17. The present study demonstrates that work
with nonglycosylated peptide substrates could principally yield
different glycosylation patterns compared with the use of glycopeptide
substrates (refer to the glycosylation patterns of A1 and A5). Hence,
the addition of GalNAc in vitro is not merely ruled by the
sequence context of acceptor sites but can be affected by preceding
glycosylation of other sites of the peptide substrate. This finding
should be considered when analyzing the specificities of
individual ppGalNAc-Ts in vitro. The use of glycosylated
peptides as substrates is justified by the assumption that in
vivo, also, the preferred target sites should be glycosylated with
GalNAc (or even with complex, core-type glycans) before initiation at
other potential, but poor substrate positions has been started.
Accordingly, the effects observed in vitro could also rule
the patterns of GalNAc addition to the mucin core peptide in the Golgi
system. We have previously shown that although all five potential sites
within the repeat peptide are glycosylation targets in vivo,
on the average, only 2.6 sites per repeat carry glycan moieties (8).
The degree of substitution at each site as revealed by quantitative
Edman degradation, in conjunction with evidence from mass spectrometry
indicate a heterogeneous pattern of site-specific glycosylation within
the repeat peptide. This heterogeneity could be explained in part by
the findings discussed above.
What sorts of conformational or steric effects could underlie the
positive or negative changes of site-specific substrate qualities? On
the basis of NMR data and molecular modeling techniques, it has
recently been suggested that ppGalNAc-Ts (T1 and T3) require at least
three points of contact to stabilize the spatial orientation of the
target hydroxyl group (18). Although one of these groups is the target
site itself, the second and third sites are located in the vicinity
(between +3/+6 and between The knowledge summarized above on peptide conformation of MUC1 at the
glycosylated motifs and on glycan/peptide interaction, in conjunction
with the suggested requirements for substrate binding by the
ppGalNAc-Ts, may partly explain why core-type glycosylation at The message of this paper is regarded to be found in a mechanistic
model that could explain the regulation of initial
O-glycosylation. The postulated dynamic regulation of
peptide glycosylation during endoplasmic reticulum/cis-Golgi
cyclization of proteins may supplement the site-specific addition of
GalNAc by specialized ppGalNAc-Ts. It remains to be established whether
glycosylation-induced effects on GalNAc transfer to particular target
sites on monorepeats of MUC1 can also be observed on multiple repeats,
because artificial effects related to the sizes of the peptide
substrates cannot be ruled out. No such effects had been observed by
Nishimori et al. (9) using nonglycosylated oligomeric MUC1
repeats. In conclusion, the differential acceptor qualities of
individual peptide positions in conjunction with enzyme repertoire and
activity of ppGalNAc-Ts in Golgi compartments should result in an
ordered sequence of glycosylation, which is partly regulated by
competitive glycosyltransferases involved in the synthesis of core-type
glycans. As presented in Fig. 3, the
density of peptide glycosylation could accordingly be regulated by the
competition between initiation by ppGalNAc-Ts and the core glycan
synthesis by or Gal
1-3GalNAc
at different positions. The
enzymatic products were analyzed by MALDI mass spectrometry and Edman
degradation for the number and sites of incorporated GalNAc.
Disaccharide placed on the first position of the diad Ser-16-Thr-17
prevents glycosylation of the second, whereas disaccharide on the
second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent
GalNAc addition to the first. Multiple disaccharide substituents
suppress any further glycosylation at the remaining sites.
Glycosylation of Ser-16 is negatively affected by glycosylation at
position
6 (Thr-10) or
10 (Ser-6) and is inhibited by disaccharide
at position
11 (Thr-5), suggesting the occurrence of
glycosylation-induced effects on distant acceptor sites. Kinetic
studies revealed the accelerated addition of GalNAc to Ser-16 adjacent
to GalNAc-substituted Thr-17, demonstrating positive regulatory effects
induced by glycosylation on the monosaccharide level. These
antagonistic effects of mono- and disaccharides could underlie a
postulated regulatory mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3GalNAc at single or multiple
positions. The site-specific activities of three recombinant enzymes
(rGalNAc-T1, -T2, and -T3) were assayed and compared with those of the
soluble enzymes shed into human milk (milk GalNAc-Ts). The products
were separated and quantitated by reversed phase HPLC, identified by
MALDI mass spectrometry and the "terminally" glycosylated peptides
with a maximum number of incorporated GalNAc were sequenced by Edman
degradation to localize the sites of glycosylation. We were able to
demonstrate that negative vicinal effects on glycosylation are exerted
on the disaccharide level and are site-restricted. On the
monosaccharide level, positive effects could be proven to occur
in vitro, resulting in greatly accelerated GalNAc transfer
to a vicinal position. Finally, also distant effects on initial
O-glycosylation were observed that suggest that
glycosylation-induced conformational changes of the peptide substrate
may influence the accessibility of particular acceptor sites for
ppGalNAc-Ts.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3GalNAc
at various
positions. Glycopeptides A11 and A13 carrying GalNAc in defined
positions of a 20-mer (HGV20) or 21-mer (AHG21) were synthesized
similarly (13). Nonglycosylated peptide TAP25, corresponding to one
repeat and five overlapping amino acids (starting with the TAP motif),
was kindly provided by Dr. Taylor-Papadimitriou (Imperial Cancer
Research Fund, London, United Kingdom).
-Thr in position 4. The corrected yield for
this cycle (repetitive yield for the glycopeptide ITATPAPT) was 95%.
The retention time of GlcNAc
-Thr was almost identical to the
corresponding GalNAc derivative on reversed phase HPLC.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Synthetic (glyco)peptides used in this
study
View larger version (24K):
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Fig. 1.
Reversed phase HPLC of reaction products
after in vitro transfer of GalNAc to glycopeptide
substrates. The ultrafiltrated reaction mixtures were applied to
PLRPS columns and run in water-acetonitrile gradients as described
under "Experimental Procedures." A, the glycopeptide
substrate A1 was incubated with the recombinant GalNAc transferases
rGalNAc-T1 (A1G1), rGalNAc-T2 (A1G2), and
rGalNAc-T3 (A1G3) and yielded the product indicated by
A1-1. B, the glycopeptide A5 was incubated with
rGalNAc-T1 (A5G1), rGalNAc-T2 (A5G2), and
rGalNAc-T3 (A5G3) and yielded the products indicated by
A5-1 and A5-2. C, the glycopeptide
substrate A11 was incubated with a mixture of the rGalNAc-Ts
(A11-G1-3) and yielded one product, indicated by
A11-1.
Number of GalNAc residues introduced into glycopeptide substrates
after 72 h of reaction time
Sites and rates of GalNAc addition to glycosylated MUC1 repeat
peptides
6 in glycopeptide
substrate A2 (Table III). The same holds true for glycopeptide A13
exhibiting monosaccharide substitution of Thr-10 (Table III). Although
the pattern of site-specific GalNAc addition on glycopeptides A2 and A13 agrees with that on the nonglycosylated control peptide TAP25, the
rate of Ser-16 glycosylation in A2 and A13 was found to be affected
negatively by glycans at Thr-10 (see kinetic analyses and Fig.
2).
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Fig. 2.
Kinetic studies of glycosylation at
individual sites using milk GalNAc-Ts The substrates were
incubated for the indicated times at 37 °C adding fresh enzyme and
cosubstrate at 24-h intervals. The products were separated and
quantified by reversed phase HPLC and identified with respect to the
number and sites of GalNAc addition by Edman degradation. The
percentages of the reaction products are shown relative to the total
amount of residual substrate and all products formed. A,
Ser-16 glycosylation in substrates A11 ( ), A3 (
), and TAP25
(
). B, Ser-16 glycosylation in substrates A2 (
), A4
(
), A13 (×), and TAP25 (
). C, Thr glycosylation of
substrates A3 (Thr-5) (
), A1 (Thr-17) (
), and TAP25 (Thr-5,
Thr-17) (
). D, kinetic of substrate decrease for A1
(
), A3 (
), and TAP25 (
). Because Thr-5 and Thr-17 are
glycosylated rapidly, the HPLC fractions corresponding to intermediate
products of several substrates (A2, A4, A13, TAP25) represent mixtures
of monoglycosylated peptides with different site substitution, and
accordingly, the kinetic of glycosylation of individual sites could not
be analyzed in these cases.
Glycosylation-induced effects on the activities of rGalNAc-T1 to -T3 as
revealed by the apparent kinetic constants for glycopeptide substrates
Extent of product formation by rGalNAc-T1 to -T3 using the glycopeptide
substrates A1, A3, and A5
6,
10, and
11 relative to the target Thr/Ser (Fig. 2, A and
B). Relative to the nonglycosylated control peptide TAP25,
all substrates with GalNAc substitution at Thr-10 (A13),
Gal
1-3GalNAc substitution at Thr-10 (A2) and Thr-5 (A1), or Ser-6
(A4) exhibit reduced (A2, A4, and A13 in Fig. 2B) or even no
detectable glycosylation of Ser-16 (A1, not shown). Compared with the
nonglycosylated control peptide TAP25, Thr-17, also, is affected
negatively by the disaccharide in Thr-5 (A1), because even after
72 h, more than 60% of substrate was detected in the reaction
mixture (Fig. 2, C and D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2/
6). It was further suggested that five
or six residues adjacent to both sides of the target Thr/Ser should be
in an extended
-like or an inverse
-turn conformation, which was
proven to exist in the VTSA motif (18). Already previous studies on
model peptides had shown that in vitro
O-glycosylation of potential target sites was dependent on a
random-coil conformation of the peptide substrate (4). Native mucins
like those from submaxillary glands tend to adopt such random-coil
structure leading to extended conformations with peptide dimensions
about 3-fold more expanded than found for deglycosylated mucins (19).
However, apomucins from submaxillary glands, also, provably exist in a
more extended, nonglobular conformation with high content of aperiodic
structure and
-turns facilitating addition of GalNAc to the peptide
scaffold (20). Nonglycosylated (21) and glycosylated VNTR peptides
of MUC12,3 have been analyzed by measurement of circular
dichroism2 and by NMR
spectroscopy,2,3 and they
turned out to form a left-handed poly-L-proline II helix stabilized by addition of GalNAc residues.2 This type of
secondary structure is a characteristic feature of mucins, because also
for other members of this subclass of glycoproteins, like the human
salivary mucin MG2, the existence of a significant population of
poly-L-proline II-type helices has been demonstrated in
aqueous solution (22). Of particular significance in the context of
this study are interactions between the sugar substituent and the
peptide backbone with influences on the peptide conformation and
substrate qualities of potential O-glycosylation sites. NOE
connectivities between the N-acetyl NH proton of the GalNAc
and the
H protons of the prolines at +3 indicate that the GalNAc
residues are positioned along the peptide backbone of MUC1 repeats in a
C-terminal direction (1). This finding is in partial agreement with
work on model glycopeptides for antifreeze glycoprotein (23) and ovine
submaxillary mucin (19). In both studies, the authors were able to
demonstrate the existence of an intramolecular hydrogen bond between
the amide proton of GalNAc and the carbonyl oxygen of the glycosylated
threonine resulting in a C-terminal orientation of the sugar relative
to the protein backbone. NMR analyses of the glycopeptides used in this
study are still in progress, but the available data confirm a
relatively extended conformation of the VNTR peptide disrupted by
polyproline helical elements and a turn-like self-stabilizing PDTR
motif.3
6
relative to the Ser-16 target site (A2, A13) negatively influences
GalNAc addition at this position (see Fig. 2B). Similar considerations should be relevant in the context of GalNAc addition to
Thr-17 in A5 and Thr-17/Thr-18 in A5b, where the Thr at +1 remains
unglycosylated, whereas that at +2 is a target for the respective
ppGalNAc-T. On the basis of available data, no straightforward explanation can be given to the negative effect exerted by
Gal
1-3GalNAc at Thr-5 on the glycosylation at Ser-16, in
particular, because no such effect was found with GalNAc substituted at
Thr-5. While long range interactions between the VTSA motif and the
PDTR motif were not observed (18), information on interactions of
distant motifs with the GSTA motif is lacking. With concern to the
positive effect on Ser-16 glycosylation exerted by GalNAc at Thr-17, it may be allowed to speculate that the sugar interaction with the proline
residue at +3 reduces the flexibility of the peptide chain and in this
way may stabilize a particular conformation that is preferred by the
transferase. Alternatively, it cannot be excluded that the binding site
of the enzyme forms specific contacts with the hydroxyl groups of sugar
residues at position +1.
-6-glucosaminyltransferase (core2 enzyme) or
-3-galactosyltransferase (core1 enzyme). Based on this postulated
regulatory model, carcinoma cells with a deficient glycosylation
machinery lacking one of these core enzymes or expressing these at low
activity levels (Fig. 3B) should glycosylate peptides to a
higher degree of substituted sites compared with normal cells (Fig.
3A). This assumption has recently been confirmed for the breast cancer cell line T47D, which does not express a functional core2
enzyme, by showing that on the average, 4.8 of the five putative sites
within VNTR peptide are
glycosylated.4 This finding
is in striking contrast to that obtained for the lactation-associated
glycoform, where only half of the putative sites per repeat had been
found to be glycosylated on the average (8). A similar relationship
between density of O-glycosylation and the chain lengths of
glycans has recently been demonstrated by structural analysis of
porcine submaxillary mucin (24).
View larger version (26K):
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Fig. 3.
Regulatory model for the initiation of
O-glycosylation on the basis of MUC1-VNTR
peptide. A relationship between rate of glycan synthesis and
glycosylation density is proposed on the basis of enzymatic competition
(core enzymes versus ppGalNAc-Ts) and the reported
glycosylation-induced effects on initial O-glycosylation.
A, O-Glycosylation of MUC1 VNTR peptide,
characterized by a low degree of site substitution with
polylactosamine-type chains; B, O-glycosylation
of MUC1 VNTR peptide, characterized by a high density of core-type
glycans.
Further studies should show on which level of glycosylation the
initiation by ppGalNAc-Ts is inhibited most effectively and whether the
postulated model has relevance also for the glycosylation of other
mucin peptides. In particular, the occurrence of positive stimulatory
effects on initial O-glycosylation that were shown to be
induced by prior GalNAc addition could explain the formation of
clustered O-linked glycans on mucins, in general. Similar positive effects exerted by prior glycosylation on poor substrate positions as
in the MUC1 model were not described yet for other mucin peptides. However, the present study calls the hypothesis into question that such
effects could explain the discrepancies found between site-specific
glycosylation of MUC1 VNTR peptide in vitro and in
vivo. Results from in vitro glycosylation (25-27) and
the derived rules on how sequences that flank Ser/Thr target sites
influence the enzyme binding to substrate have to be critically
re-evaluated. Site-specific glycosylation patterns predicted on the
basis of in vivo data (28) agree better with the actual
localization of O-linked glycans.
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ACKNOWLEDGEMENT |
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We thank Dr. F. Hillenkamp (University of Münster, Germany) for kind permission to use the VISION 2000 instrument for the performance of MALDI mass spectrometry.
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FOOTNOTES |
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* This investigation was supported by the Deutsche Forschungsgemeinschaft Grant Ha2092/4-2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-221-478-4493; Fax: 49-221-478-6977; E-mail: franz.hanisch{at}uni-koeln.de.
2 D. I. R. Spencer, S. Missailidis, C. DeMatteis, M. S. Searle, S. J. B. Tendler, and M. R. Price, presented at the Workshop on Mucin O-Glycosylation: Sites and Processing, Copenhagen, Denmark 1997.
3 J. Dojahn, C. Diotel, H. Paulsen, and B. Meyer, B., presented at the 5th International Workshop on Carcinoma-associated Mucins, Cambridge, 1998, and personal communication by Hans Paulsen.
4 S. Müller, K. Alving, J. Peter-Katalinic, N. Zachara, A. A. Gooley, and F.-G. Hanisch, submitted for publication, and presented at the 5th International Workshop on Carcinoma-associated Mucins, Cambridge, 1998.
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ABBREVIATIONS |
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The abbreviations used are: GalNAc, N-acetylgalactosamine; rGalNAc-T, recombinant polypeptide:N-acetylgalactosaminyltransferase; ppGalNAc-T, polypeptide:N-acetylgalactosaminyltransferase; Gal, galactose; MALDI, matrix-assisted laser desorption ionization; HPLC, high performance liquid chromatography.
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