(Received for publication, May 31, 1995)
From the
Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E. P., Takio, K., Sørensen, T., Bonding, N., and Clausen, H.(1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the GalNAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separated transferase preparations was identified. Analysis of GalNAc-transferase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the existence of multiple GalNAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of GalNAc-transferases existing is unknown, as is the specificity of the until now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme proteins. The identification of acceptor peptides that can be used to discriminate GalNAc-transferase activities is an important step toward understanding the molecular basis of GalNAc O-linked glycosylation in cells and organs and in pathological conditions.
Glycosylation of proteins in eukaryotes is fundamental for the integrity of the individual cell and the organism as a whole (Varki, 1993). A number of different types of protein glycosylations have been identified (for a recent review see Lis and Sharon, 1993). Biosynthesis of the initial glycosylation of the protein backbone has been established in most cases and the involved glycosyltransferases partly characterized. In several cases characterization of glycosylation sites has identified peptide motifs that suggest the nature of the acceptor substrate specificities of transferases initiating protein glycosylation. Thus N-linked asparagine glycosylation is restricted to the sequence -Asn-Xaa-Ser/Thr- (where Xaa may be any amino acid except proline). Proteoglycan-type glycosylation of serine is restricted to -Ser-Gly-Xaa-Gly- (Bourdon et al., 1987). The GlcNAc-type glycosylation of serine or threonine appears to be adjacent to an acidic amino acid and within two residues of a proline (Haltiwanger et al., 1992). The fucose-type glycosylation of serine/threonine seems to be restricted to the peptide sequence -Gly-Gly-Thr/Ser-Cys-, although the enzyme has yet to be characterized (Harris and Spellman, 1993).
In contrast, a defined peptide motif
for GalNAc O-glycosylation (mucin type) and the equivalent
yeast Man-type glycosylation of serine/threonine has not emerged. A
number of studies have attempted to identify a consensus sequence for
mammalian GalNAc O-glycosylation by studying sequences around
identified glycosylation sites (Gooley et al., 1991;
O'Connell et al., 1991; Wilson et al., 1991;
Elhammer et al., 1993) as well as by testing the peptide
substrate specificity of the GalNAc-transferase ()activity
in crude and pure form (O'Connell et al., 1992; Wang et al., 1992, 1993; Elhammer and Kornfeld, 1986; Hagen et
al., 1993; O'Connell and Tabak, 1993; Gooley and Williams,
1994; Nishimori et al., 1994a, 1994b). It is clear from these
studies that the GalNAc-transferase must have broad acceptor substrate
specificity, but it is likely that our understanding of this broad
motif is shadowed by the involvement of several GalNAc-transferases. As
described in the accompanying paper (White et al., 1995) a
novel GalNAc-transferase has been isolated and cDNA cloned, which,
together with the previously cloned bovine GalNAc-transferase (Homa et al., 1993), clearly establishes the existence of at least
two distinct enzymes. By analogy, Strahl-Bolsinger et al. (1993) provided evidence that more than one polypeptide O-mannosyltransferase exists in yeast.
The total number of existing GalNAc-transferases is unknown, but it is very likely that our knowledge of this family of transferases will expand rapidly. Assigning detailed acceptor substrate specificity toward the cloned and expressed GalNAc-T1 and -T2 awaits comparative studies of recombinant transferases, and data obtained so far using purified enzyme preparations are likely to be biased by copurified mixtures of enzymes (Homa et al., 1993; White et al., 1995; Wang et al., 1993). To begin to understand the potential differential specificity of multiple GalNAc-transferases we have begun searching for acceptor substrate peptides that would show differences as acceptors for different GalNAc-transferase preparations.
Here we present evidence that affinity chromatography using a defined synthetic acceptor substrate peptide resulted in separation of two different GalNAc-transferase activities and that these appear to be differentially expressed in organs. The results thus provide the first evidence for the involvement of at least two GalNAc-transferase specificities in GalNAc O-linked glycosylation initiation. The study identified acceptor peptides capable of discriminating different GalNAc-transferase activities, which should prove valuable for detailed studies of the specificity of identified and cloned enzymes in this family.
When measuring K and V
for different peptide
substrates the UDP-[
C]GalNAc concentration was
increased to 0.2 mM (4,000 cpm/nmol), and the enzyme
concentration was 0.5 milliunits/ml.
Preparative glycosylation of
peptides was performed with 0.5 µmol of peptide, 5 milliunits of
enzyme, and 5-100 µmol of
UDP-[C]GalNAc in a final volume of 1 ml. The
glycopeptide was purified by C-18 reverse phase chromatography, and the
glycopeptide-containing fractions were detected by scintillation
counting.
All mass spectra were obtained on a Bruker reflex Time of Flight mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany). Data were acquired by a LeCroy 9450A 400 megasamples/s digital storage oscilloscope (LeCroy Corporation, Chestnut Ridge, NY) from which single shot spectra were transferred to a MacIntosh Quadra 950 computer (Apple Computer Inc., Cupertino, CA) via a National Instruments NI DAQ GPIB controller board (National Instruments, Austin, TX).
Control of data acquisition parameters, the transfer and subsequent averaging of spectra, as well as further data processing were carried out using the computer program LaserOne, which was written in ThinkC (Symantec Corporation, Cupertino, CA) by M. Mann and P. Mortensen, EMBL, Heidelberg, Germany.
All mass spectra were obtained in the linear mode and calibrated using a singly charged matrix ion, which provided a mass accuracy of approximately 0.1%.
In some experiments Triton X-100 was exchanged at the S-Sepharose step (step 3) by slow overnight washing with n-octyl glucoside and maltoside, and further purification was performed in n-octyl glucoside-containing buffers that were otherwise as described.
The purification scheme used gave a quantitatively different result for the submaxillary gland activity compared with human placenta. The initial Cibacron chromatography of ovine and porcine gland extracts yielded the same results as for the human placenta transferase. However, the yields of the ovine and porcine transferases were considerably lower at the peptide affinity chromatography step compared with human placenta. Since the human transferase purified by the same procedure using Triton X-100 as detergent in the affinity chromatography was found to be the soluble fragment without the hydrophobic transmembrane segment, this difference in yield could be related to a relatively lower ratio of soluble versus membrane-bound transferase. Human placenta tissue was obtained 6-24 h after delivery (stored at room temperature) and after freezing was subsequently thawed at 4 °C for 1-3 days before extraction. In contrast the animal glands were quick frozen by the supplier (Pel-Freez) and thawed at 4 °C overnight.
In separate experiments (not shown) the unbound fraction of the Muc2 affinity chromatography of porcine gland enzyme was reapplied to the Muc2 affinity column after detergent exchange from Triton X-100 to n-octyl glucoside and maltoside. Detergent exchange of the nonretained material from the Muc2 peptide column using n-octyl glycoside and maltoside followed by repeated Muc2 peptide affinity chromatography run in these detergents resulted in a considerably higher purification yield, although even repeated application on the column failed to absorb more than 50% of the transferase activity measured with the Muc2 peptide substrate. The human placenta preparation behaved similarly except for the initially higher yield of the first chromatography run in Triton X-100. Ion exchange Mono S chromatography performed without detergent on the porcine and ovine preparations obtained after detergent exchange and Muc2 affinity purification resulted in total loss of activity. Gel filtration of the same preparations gave a spread of enzyme activity from the void volume to a molecular weight of approximately 100,000 (not shown), all suggestive that the transferases were bound tightly to the detergent and therefore likely to include a strongly hydrophobic transmembrane segment.
Figure 1:
K determination of ovine GalNAc-transferase at different
stages of purification. Ovine GalNAc-transferase was purified on the
Muc2 peptide affinity column (step 5). K
values were measured as described under ``Experimental
Procedures'' and calculated from a Lineweaver-Burk double
reciprocal plot to 231, 254, and 50 µM for the precolumn
preparation (S-Sepharose eluate, step 4), the nonretarded material, and
the eluate, respectively.
Analysis
of the substrate specificity of enzyme preparations before the Muc2
affinity purification, the unbound flow-through fraction, and the bound
and eluted enzyme preparations from the column using various synthetic
peptide substrates is presented in Table 3and Fig. 2.
Human placenta and ovine and porcine submaxillary transferases in the
Muc2 affinity-purified form all contained both threonine and serine
transferase activity as measured by the human chorionic
gonadotropin- peptide, which has only serine acceptor sites,
although with a very low activity. Strikingly, all of the Muc2
affinity-purified transferase preparations (step 4) failed to
glycosylate the HIV-V3 peptide sequence, whereas the enzyme
preparations before affinity chromatography as well as the nonretarded
materials from the affinity chromatography column readily utilized this
substrate. The product of the HIV-V3 peptide glycosylated by pre-Muc2
affinity-purified enzyme preparations (step 1 or 3) was confirmed as
containing a single GalNAc residue attached to the single Thr in an
undegraded peptide by amino acid sequencing (Fig. 3) and mass
spectrometry (not shown). Although the HIV-V3 peptide is an acceptor of in vitro enzymatic glycosylation it is not known if indeed
this site serves as an in vivo O-glycosylation site. HIV gp120
is, however, O-glycosylated, and GalNAc-Ser/Thr epitopes have
been identified (Hansen et al., 1992; Merkle et al.,
1991).
Figure 2:
Competition among different acceptor
peptide substrates using human placenta GalNAc-transferase activities
at different purification stages. Incorporation of
[C]GalNAc into the different peptides was
analyzed by C-18 reverse phase separation of products and scintillation
counting of peaks corresponding to the individual glycopeptides.
Competitive assays were performed in the presence of 0.2 mM
UDP-[
C]GalNAc. Panel A, HIV-V3 peptide versus Muc2 peptide using S-Sepharose-purified enzyme (step
4). A competitive transferase assay was performed with a constant
amount of HIV-V3 peptide (1 mg/ml) and an increasing amount of Muc2
peptide. Incorporation of GalNAc into HIV-V3 peptide decreased with an
increasing amount of Muc2, indicating that the peptides were substrates
for the same enzyme. Panel B, Muc1 peptide versus Muc2 peptide using Muc2 affinity-purified enzyme (step 5). A
competitive transferase assay was performed using a constant amount of
Muc1 (150 µg/ml) and an increasing amount of Muc2. Incorporation of
GalNAc into Muc1 peptide decreased with an increasing amount of Muc2,
indicating that the peptides were substrates for the same enzyme. Panel C, Muc2 peptide versus HIV-V3 peptide using
Muc2 affinity purified enzyme (step 5). A competitive transferase assay
was performed using a constant amount of Muc2 (150 µg/ml) and an
increasing amount of HIV-V3. Incorporation of GalNAc into Muc2
decreased with increasing amounts of HIV-V3 without incorporation of
GalNAc into HIV-V3. For comparison, Muc2 peptide versus HIV-V3
peptide using S-Sepharose-purified enzyme (step 4) showed no
significant effect on the incorporation into
Muc2.
Figure 3: Amino acid sequencing of the glycosylated HIV-V3 peptide. The HIV-V3 peptide was terminally glycosylated as described under ``Experimental Procedures.'' The GalNAc glycosylated threonine is ``seen'' as a markedly reduced threonine peak in the cycle corresponding to this amino acid compared with the amount of amino acid in the adjacent amino acid cycles. The upper, middle, and lower panels show the cycles corresponding to valine, threonine, and isoleucine, respectively. Note the pseudopeaks with a retention time of 9.67 close to the Gln-phenylthiohydantoin derivative.
To exclude that this difference could be ascribed to soluble versus membrane forms of enzymes tested, the apparently
membrane-bound form of the GalNAc-transferase was purified and analyzed
after detergent exchange. As shown in Table 3the difference in
substrate specificity was consistent also for the porcine transferase
purified after detergent exchange. Several peptides with sequences
overlapping the HIV-V3 peptide were analyzed to exclude a specific
problem with the peptide design of HIV-V3, and all peptides showed the
same reaction pattern with the transferase preparations tested. In Table 3only the peptide HXB2 with the same internal sequence as
HIV-V3 but with extended sequence in both NH and COOH
termini is shown.
The HIV-V3 acceptor substrate peptide was
identified by application of 15-mer overlapping peptides covering the
entire sequence of the HIV envelope protein gp120 as
acceptor substrate in the GalNAc-transferase assay in an attempt to
predict O-linked glycosylation sites by in vitro enzyme assay (Clausen et al., 1994). Subsequent analysis
of these peptide sequences by the prediction model of Elhammer et
al.(1993) has shown that the glycosylation probability of the
HIV-V3 peptide sequence is 0 and is therefore not predicted by the
model.
To characterize the different transferase activities further, competitive glycosylation experiments were performed. Since the GalNAc-transferase preparation before the Muc2 affinity chromatography was capable of glycosylating the HIV-V3 peptide as well as the Muc2 peptide, it was pertinent to analyze if one enzyme utilized both of these substrates. As shown in Fig. 2A the Muc2 peptide was a competitive inhibitor of HIV-V3 peptide glycosylation suggestive of one enzyme utilizing both substrates. Control experiments using Muc1 and Muc2 peptide with Muc2 affinity-purified transferase also showed competitive inhibition of Muc2 glycosylation (Fig. 2B). Surprisingly, the HIV-V3 peptide, which was not glycosylated by the Muc2 affinity-purified transferase, was found to be an inhibitor of Muc2 glycosylation using this enzyme preparation. Adding increasing amounts of HIV-V3 peptide to Muc2 affinity-purified transferase showed competitive inhibition of Muc2 glycosylation. These results suggest that Muc2 peptide affinity chromatography separates two distinct transferase activities with overlapping specificity concerning the Muc2 acceptor substrate but which are distinguishable with respect to the HIV-V3 peptide.
As described
under ``Experimental Procedures'' the GalNAc-transferase
preparations used were partially purified either to step 1 or to step 3
(inclusive). As such transferase preparations may include interfering
proteolytical activity, the following steps were taken to exclude this.
1) Routine assays included identification of product by Dowex 1
chromatography, but in all combinations of peptide and enzyme
preparations C-18 chromatography of the reaction mixture revealed the
same UV profile. Potentially incorporated
[C]GalNAc was always found associated with the
peptide peak (only when peptides containing multiple acceptor sites
were used, were products clearly separable from the unglycosylated
peptide peak by the conditions used). 2) Mass spectrometric analysis of
substrate peptides after prolonged incubation with one of the organ
enzyme preparations (human liver, purification step 1) showed no
evidence of degradation (Fig. 4). 3) Sequential incubation of
acceptor substrate peptides first with an organ enzyme preparation not
capable or only poorly capable of incorporating GalNAc (human liver,
purification step 1) and second with an organ enzyme preparation
capable of incorporating GalNAc (human placenta, purification step 3)
showed little or no loss/decreased accessibility of acceptor substrates (Fig. 5).
Figure 4:
Mass spectrometry analysis of
acceptor-substrate peptides during incubation with crude
GalNAc-transferase preparations. Human liver enzyme (purification step
1) and the reaction mixture including acceptor substrate peptides were
incubated at 37 °C; aliquots were taken at 0, 60, and 120 min, and
6 and 24 h. These aliquots were passed through a Dowex 1 column, and
the pass-through was used for measuring the incorporation of
[C]GalNAc as well as for mass spectrometry.
Spectra of HIV-V3 and Muc1a peptide are shown for 0 and 120 min. There
is no significant fragmentation of the two peptides during this time
interval. Both peptides contain a terminal cysteine amino acid and
therefore easily form dimers, which are seen in the spectra. Reduction
in the amount of dimer is due to the presence of 2-mercaptoethanol in
the reaction mixture. The human liver enzyme was eluted from the
Cibacron column with 1.5 M KC1 (purification step 1) and used
directly in the assays. This may account for the strong cationization
of the peptide with two K
ions and the peak
corresponding to (M-H+2K
) being dominant.
Surprisingly, there is no cationization of the dimer, and at present we
have no explanation for this finding.
Figure 5:
Sequential enzyme assays to monitor the
stability of acceptor substrate peptides. Peptides Muc1a and HIV-V3
were incubated at 37 °C with human liver enzyme (purification step
1) in the standard reaction mixture for various times. Immediately
after each time interval human placenta (purification step 4) and
UDP-[C]GalNAc were added and incubated as
described for the standard polypeptide GalNAc-transferase assays under
``Experimental Procedures.'' The ability of the placenta
enzyme to glycosylate the peptides after incubation with the liver
enzyme as a percentage of the initial ability at t = 0
is presented.
In the accompanying paper (White et al., 1995) we
describe the purification of a human GalNAc-transferase using a defined
acceptor substrate peptide as a major affinity ligand. During the
purification work we found that only a small fraction of enzyme
activity was bound to the column even upon repeated chromatography,
suggesting that possibly different transferase activities were present.
Further support for this hypothesis was found by an observed lower K value toward the peptide used (Muc2) for the
affinity chromatography in the Muc2 affinity-purified preparation
compared with the enzyme preparation immediately before this step and
the enzyme preparation that passed through the column. Clearer evidence
for the existence of two GalNAc-transferase activities was found by
analyzing the acceptor substrate specificity of these enzyme
preparations at different stages of the purification. This resulted in
the identification of a qualitative difference in glycosylation of a
HIV gp120 peptide. This peptide was an excellent substrate during the
early steps of purification of the human placenta enzyme as well as the
enzyme preparation that passed through the Muc2 affinity column (step
4), whereas the Muc2 affinity-purified enzyme lacked such activity.
This phenomenon was found to be species-independent as both ovine and
porcine submaxillary gland Muc2 affinity-purified transferase showed
the same pattern of activity.
In the course of this work two
independent groups have reported the isolation and cloning of a bovine
GalNAc-transferase (GalNAc-T1) (Homa et al., 1993; Hagen et al., 1993) that is different from the human
GalNAc-transferase reported in the accompanying paper (GalNAc-T2)
(White et al., 1995), thus establishing that at least two
members of this transferase family exist. The fine specificity of these
two GalNAc-transferases has yet to be established in a comparative
study of recombinant expressed enzymes. The present results show
independently that two distinct transferase activities may be
recognized, and substrates capable of distinguishing these have now
been identified. Preliminary data suggest that neither human GalNAc-T1
nor GalNAc-T2 utilizes the HIV-V3 peptide (soluble constructs expressed
in a baculovirus system). ()
The presented data clearly
establish that at least two distinct GalNAc-transferase activities can
be identified and separated. The specificity of these activities
appears to be overlapping to a large extent as evidenced by competitive
substrate analysis (Fig. 2). The competitive inhibitor effect of
the HIV-V3 peptide on the Muc2 affinity-purified GalNAc-transferase
indicates that the purified transferase recognizes and binds the HIV-V3
peptide but cannot transfer GalNAc to it. Inhibition of glycosylation
by nonglycosylating peptides has been noted previously by
O'Connell et al.(1992). In addition, an observed de
novo appearance of GalNAc-transferase activity during the
purification of UDP-Gal:GalNAc-Ser/Thr
1-3-galactosyltransferase may be relevant to this
(Brockhausen et al., 1992). Analysis of the substrate
specificity of impure GalNAc-transferase preparations may thus be
biased by inhibiting factors. The observed difference in specificity of
the separated GalNAc-transferase activity may be associated with a
cofactor/modulator as found for UDP-Gal GlcNAc
1-4-galactosyltransferase (McGuire et al., 1965);
however, several lines of evidence indicate this is not the case. First
is the finding that the GalNAc-transferase activities can be physically
separated yielding two activities with the purified activity having
apparent lower K
and otherwise broad specificity.
Second, transferase activity with a specificity similar to that of the
purified transferase is found in crude extracts of certain organs (Table 4). Finally, to date two distinct GalNAc-transferase
proteins have been isolated and cDNA cloned, and these show
differential organ distribution by Northern analysis (White et
al., 1995; Homa et al., 1993).
Detailed understanding
of the acceptor-substrate specificity of different GalNAc-transferases
clearly has to await cloning and expression of all members of this
family of enzymes. It was previously expected that a difference in
substrate specificity of multiple enzymes could be related to Ser and
Thr acceptor sites (Wang et al., 1992; O'Connell et
al., 1992; Harada et al., 1985); however, purified
GalNAc-transferase from bovine colostrum has been recently shown to
exhibit specificity for both (Homa et al., 1993). The present
data corroborate this for GalNAc-T2, showing a near proportional
purification of both Thr and Ser activities (Table 3), and
recombinant expressed GalNAc-T2 also showed Ser activity (White et
al., 1995). Recently, Wang et al.(1992) showed that
purified porcine submaxillary gland GalNAc-transferase, which is
reported to be identical to GalNAc-T1 (Roth et al., 1994),
exhibits very high substrate specificity for the human erythropoietin
sequence -Ala-Ala-Ser-Ala-Ala-. Our preliminary data indicate that
recombinant GalNAc-T1 is devoid of such activity, and recombinant
GalNAc-T2 is very poor in utilizing this substrate. ()The
reason for these discrepancies are presently unknown but are under
study.
In the present study we have worked primarily with acceptor peptides derived from proteins with unknown in vivo O-glycosylation patterns. The peptides derived from human mucin tandem repeats are likely to be glycosylated in vivo at least partly, but for the HIV and SIV peptides it is only known that a few O-glycosylation sites are utilized in vivo on these large glycoproteins (Hansen et al., 1992; Merkle et al., 1991). Recently, Elhammer et al.(1993) proposed a prediction model for O-glycosylation based on the occurrence of amino acid residues positioned ±4 to identified O-glycosylation sites. The prediction model does not identify the HIV-V3 sequence. However, a comparison of the prediction model with an in vitro GalNAc-transferase assay using 32 15-mer peptides covering the entire HIV gp120 protein allowed identification of three out of four sites by both methods; additionally, three sites were identified only by the in vitro enzyme assay (Clausen et al., 1994). Thus, some correlation was found between the statistically predicted sites and in vitro glycosylation, but the in vitro glycosylation assay using crude GalNAc-transferase (Cibacron eluates corresponding to step 1 in Table 1) identified additional sites. Whether these sites indeed are glycosylated in vivo is under study, but difficulties in obtaining pure viral envelope proteins in sufficient quantity for structural analysis have hampered this effort. Importantly, the in vitro GalNAc-transferase assay identified sites in the hypervariable V3 loop of different HIV and SIV isolates, and O-glycosylation could therefore mask this principal neutralizing epitope. In fact, the HIV-V3 sequence has been shown to contain a T-cell class I epitope, and the predicted glycosylation sites are positioned in the middle, thus presumably being able to mask the site (Ishioka et al., 1992; Mouritsen et al., 1994).
Our analysis of the acceptor-substrate specificity of GalNAc-transferase preparations from different organs demonstrated both quantitative and qualitative differences (Table 4). A number of control experiments ruled out that these differences in specificity were a result of degradation and/or unknown blocking of acceptor substrate peptides. The most striking finding was that the HIV-V3 peptide indicated a qualitative distinction between enzyme extracts, thus agreeing with our interpretation that the Muc2 affinity chromatography results in the separation of two distinct GalNAc-transferase activities.
The differential organ expression of
GalNAc-transferase activities using HIV-V3 peptide was further
corroborated by analysis of two partial sequences of the Muc1 20-mer
tandem repeat (Table 4). Interestingly, the ability to
glycosylate HIV-V3 correlated with Muc1a glycosylation and lack of
HIV-V3 enzyme activity with Muc1b glycosylation. Northern analysis of
GalNAc-T1 (Homa et al., 1993) and GalNAc-T2 (White et
al., 1995) expression indicated that human kidney and liver
preferentially express GalNAc-T2, and these organs (rat kidney, human
liver) appear to express a substrate specificity in agreement with that
found for GalNAc-T2. The finding of apparent differential organ
glycosylation of the in vitro identified glycosylation sites
in the Muc1 tandem repeat (Muc1a: -Thr-Ser-; Muc1b: -Ser-Thr-) may be
important for understanding the molecular basis of cancer-associated
epitopes mapped to the Muc1 tandem repeat (Gendler et al.,
1990). A number of antibodies to Muc1 have been generated, and most of
these map to the knob-like structure defined by
-Ala-Pro-Asp-Thr-Arg-Pro- (Taylor-Papadimitriou et al., 1993).
Flanking these repeated knobs are the -Thr-Ser- and -Ser-Thr- motifs,
which the present study indicates are differentially glycosylated by
independent GalNAc-transferases. Structural analysis of in vitro glycosylated Muc1 tandem repeats using breast and pancreatic cell
line extracts as well as semipurified human placenta GalNAc-transferase
(purification steps 1 and 3) indicates that only the flanking sites are
glycosylated and that the single Thr in the knob tip
(-Pro-Asp-Thr-Arg-) is left unglycosylated (Nishimori et al.,
1994a, 1994b). ()If the observed difference in in vitro glycosylation reflects the in vivo processing this
finding may have implications for the structure of Muc1 expressed in
different organs and in cancer cells.
In conclusion, the present study provides evidence that different GalNAc-transferase activities are involved in the initiation of GalNAc O-glycosylation and that these are differentially expressed in cells and organs. Identification of suitable acceptor substrates capable of distinguishing such transferase activities is believed to be a significant step forward in the understanding of GalNAc O-glycosylation processing and will be valuable for characterization of the substrate specificity of different GalNAc-transferase genes as these are cloned and expressed.