(Received for publication, November 13, 1996, and in revised form, March 27, 1997)
From the Departments of Medical Chemistry and
§ Experimental Zoology, Vrije Universiteit,
1081 BT Amsterdam, The Netherlands
Lymnaea stagnalis
UDP-GlcNAc:GlcNAc-R
1
4-N-acetylglucosaminyltransferase (
4-GlcNAcT) is
an enzyme with structural similarity to mammalian UDP-Gal:GlcNAc
-R
1
4-galactosyltransferase (
4-GalT). Here, we report that also
the exon organization of the genes encoding these enzymes is very
similar. The
4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons)
contains four exons, encompassing sequences that are absent in the
4-GalT gene. Two of these exons (exons 7 and 8) show a high sequence
similarity to part of the preceding exon (exon 6), suggesting that they
have originated by exon duplication. The exon in the
4-GalT gene,
corresponding to
4-GlcNAcT exon 6, encodes a region that has been
proposed to be involved in the binding of UDP-Gal. The question
therefore arose, whether the repeating sequences encoded by exon 7 and
8 of the
4-GlcNAcT gene would determine the specificity of the
enzyme for UDP-GlcNAc, or for the less preferred UDP-GalNAc. It was
found that deletion of only the sequence encoded by exon 8 resulted in
a completely inactive enzyme. By contrast, deletion of the amino acid
residues encoded by exons 7 and 8 resulted in an enzyme with an
elevated kinetic efficiency for both UDP-sugar donors, as well as for
its acceptor substrates. These results suggest that at least part of
the donor and acceptor binding domains of the
4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and
specificity.
Glycosyltransferases form a large family of functionally related,
membrane-bound enzymes that are involved in the biosynthesis of the
carbohydrate moieties of glycoproteins and glycolipids (1, 2). Recently
we have identified a novel glycosyltransferase by the isolation of a
UDP-GlcNAc:GlcNAc-R
1
4-N-acetylglucosaminyltransferase (
4-GlcNAcT)1 cDNA from the prostate
gland of the snail Lymnaea stagnalis (3). In
vitro, the recombinant
4-GlcNAcT catalyzes the transfer of GlcNAc from UDP-GlcNAc in
1
4 linkage to various
-N-acetylglucosaminides (3, 4). The
4-GlcNAcT cDNA
appeared to show a significant sequence similarity to the mammalian
UDP-Gal:GlcNAc
-R
1
4-galactosyltransferase (
4-GalT)
cDNAs, with an overall resemblance between the predicted amino acid
sequences of about 30% (3, 5-7). Based on the genetic and enzymatic
relationship of the
4-GlcNAcT and the
4-GalTs, we have suggested
that these enzymes constitute a separate glycosyltransferase gene
family, the members of which are capable of catalyzing the transfer of
a specific sugar from their respective UDP-sugar donors in a
1
4
linkage toward a terminal
-linked GlcNAc residue in the acceptor (3,
8). Based on enzymatic properties, we have proposed that also
UDP-GalNAc:GlcNAc
-R
1
4-N-acetylgalactosaminyltransferase (
4-GalNAcT),
detected in several non-vertebrate species (9-13), belongs to this
family (14). The primary structure of this enzyme, however, is still
unknown.
The reaction catalyzed by glycosyltransferases typically involves two
substrates and often a divalent cation cofactor. This suggests that the
enzymes consist of several functional domains involved in substrate and
cofactor binding, respectively. Comparison of the conserved and
variable regions of genetically related glycosyltransferases with
different properties would open possibilities to address structure-function relationships. As snails and mammals are
evolutionary distant species, comparison of the genomic organization of
the genes that encode these enzymes might give insight in their way of
divergence, that resulted in genes encoding enzymes with a different
UDP-sugar specificity. The genomic organization of the murine and human
4-GalT genes have been described previously (15, 16). Here we report
the organization of the L. stagnalis gene that codes for the
4-GlcNAcT. The intron-exon distribution was determined and compared
with that of the
4-GalT gene. Mutant
4-GlcNAcT cDNAs were
constructed by deletion of sequences that do not have a counterpart in
the
4-GalT gene, and expressed in COS cells. Comparison of the
kinetic parameters of the resulting mutant and parental enzymes showed
that the insertion in the
4-GlcNAcT and its surrounding regions
contribute to acceptor recognition as well as to UDP-sugar binding and
specificity.
Acceptor substrates were obtained from Sigma (compounds 1, 2, and 3) and from Toronto Research Chemicals (compounds 5 and 6). Compound 4 was a gift of Dr. O. Hindsgaul, University of Alberta, Alberta, Canada. UDP-[3H]GlcNAc, UDP-[3H]GalNAc and UDP-[3H]Gal were purchased from NEN Life Science Products, and were diluted with unlabeled UDP-sugars (Sigma) to the desired specific radioactivity.
Recombinant plasmids were propagated in the Escherichia coli K12 strain XL1-Blue (Stratagene). COS-7M6 cells (ATCC 1651) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1:50 penicillin/streptomycin solution (all from Life Technologies, Inc.). Synthetic oligonucleotides were obtained from Isogen Bioscience BV (Maarssen, the Netherlands).
DNA TechniquesThe genomic clone 5 has been isolated
previously (3) from a genomic
EMBL3A library of L. stagnalis (17). Isolation of plasmid DNA was carried out by a
modification of the minilysate method, as described in Ref. 18.
Plasmids used for transfection of COS cells were isolated by the Qiagen
plasmid protocol, using a QIAGEN-tip 100 minicolumn. Restriction
enzymes and other DNA-modifying enzymes were used according to the
manufacturer. Dideoxynucleotide chain-terminating sequencing reactions
(19) were performed on double-stranded plasmid DNA, with the T7 DNA
sequencing kit (Pharmacia Biotech Inc.), [
-35S]dATP
(Amersham), using M13 universal primer, the KS and SK primers, and
several sequence-specific synthetic oligonucleotide primers. Southern
blotting was performed as described previously (3). PCR with
sequence-specific primers was performed using Ultma-polymerase (Perkin-Elmer) by 25 cycles (1 min at 95 °C, 1 min at 63 °C, 1 min at 72 °C). For cloning purposes, amplified fragments were subsequently purified according to the QIAquick PCR purification protocol (Qiagen Inc.)
The plasmid pMC135,
containing a fusion between part of the protein A sequence and
4-GlcNAcT cDNA, was constructed as follows. A
BamHI-EcoRI adapter was ligated in the
BamHI site of pVTBac-P11.4, carrying 5
-truncated
4-GlcNAcT cDNA (3). The resulting 1.4-kb EcoRI
fragment from this construct was ligated into an
EcoRI-digested pPROTA vector (20). Plasmids carrying mutant
4-GlcNAcT genes were derived from pMC135 by exchange of the 0.52-kb
XhoI-BglII fragment for a PCR fragment carrying
the desired deletion. For PCR of the deletion fragments, a sense primer
(bases 439-456 of the
4-GlcNAcT cDNA; Ref. 3) and the antisense
primer ID8 (ATAGATCTTAAATCTGTCCGGGTTCACATTCCAG) or ID16
(ATAGATCTTAAATCTGTTCGGGTGCACGTTC) was used. The antisense primer ID8, used for construction of pMC142, consists of a part complementary to the 3
end of exon 6, and a part (11 base pairs), complementary to the 5
end of exon 9 (BglII restriction
site underlined). The antisense primer ID16, used for construction of
pMC166, consists of a part complementary to the 3
end of exon 7, and
the same 5
part of exon 9 as ID8. PCR fragments obtained with these
primers were digested with XhoI and BglII, and
ligated into XhoI-BglII-digested pMC135. After
transformation and plasmid isolation of several transformants, the
desired plasmids (pMC142 encoding protA-
4-GlcNAcT
7-8 and pMC166
encoding protA-
4-GlcNAcT
8) were selected by size determination of
the internal SalI fragment followed by determination of the
nucleotide sequence of the complete XhoI-BglII
fragment with sequence-specific primers.
Recombinant pPROTA chimeric
constructs were transiently transfected to COS cells (3 × 105 cells/10-cm dish), using the calcium phosphate
precipitation technique as described previously (21); after 24 h,
the medium was replaced by fresh medium; medium was harvested at 48, 72, and 96 h after transfection and each time replaced by fresh
medium. The harvested media were pooled, stored at 20 °C in
portions, and used as enzyme source for glycosyltransferase assays and
Western blotting. Membrane bound recombinant
4-GlcNAcT was produced
with a pMT2-based construct as described previously (3).
The Western blot analysis performed was aimed on detection of the protein A part of the fusion proteins. The proteins of 1 µl of pooled medium collected after transfection, were separated by SDS-polyacrylamide gel electrophoresis on 10% gels using the Mini-PROTEAN II system (Bio-Rad). Western blotting was performed essentially as described previously (21). As first antibody, an arbitrary mouse IgG monoclonal antibody (ED3, Ref. 22) was used, which reacts with the protein A part of the hybrid proteins. The second antibody used was a goat anti-mouse peroxidase conjugate (Tago, Inc. Immunodiagnostic Reagents).
Glycosyltransferase AssaysStandard glycosyltransferase
assays were performed in a 50-µl reaction mixture containing either
25 nmol of UDP-[3H]GalNAc (2 Ci/mol),
UDP-[3H]GlcNAc (1 Ci/mol), or UDP-[3H]Gal
(1 Ci/mol), 5 µmol of sodium cacodylate buffer, pH 7.2, 1 µmol of
MnCl2, 0.2 µmol of ATP, and 50 nmol of
p-nitrophenyl-N-acetyl-1-thio--D-glucosaminide (GlcNAc-S-pNP). As enzyme source, 10 µl of COS cell medium, which had
been concentrated 10 times with Centriprep-10 concentrators (Amicon),
was used. The product was isolated using Sep-Pak C-18 cartridges
(Waters) (23). For acceptor specificity studies, acceptor substrate
concentrations were kept at 1 mM, in terms of terminal
GlcNAc residues. Control assays lacking the acceptor substrate were
carried out to correct for incorporation into endogenous acceptors.
Enzyme activity was expressed as
pmol/min
1/ml
1 of the original medium. As
enzyme source for the experiments studying the UDP-Gal specificity, the
enzyme was isolated from the medium by binding to IgG-agarose (Sigma).
10 ml of medium was incubated with 10 µl of IgG-agarose beads for
16 h at 4 °C, carefully shaking; the beads were subsequently
collected by centrifugation and resuspended in 500 µl of 0.1 M sodium cacodylate buffer, pH 7, containing 1 mg/ml bovine
serum albumin; 10 µl of this suspension was used in a standard assay.
Kinetic parameters (Km and V) were estimated from Lineweaver-Burk plots by varying the sugar-donor concentrations from 0.05 to 0.5 mM for UDP-GlcNAc and from 0.25 to 5 mM for UDP-GalNAc while keeping the GlcNAc-S-pNP concentration at 1 mM, or by varying the acceptor substrate concentration from 0.025 to 1 mM while keeping the UDP-GlcNAc concentration at 0.5 mM. The inhibitory effect of UDP was studied at fixed concentrations of UDP-GlcNAc (0.5 mM) and GlcNAc-O-pNP (1 mM), whereas the UDP concentration was varied from 0.1 to 5 mM.
Product CharacterizationProduct, obtained with the enzyme
protA-4-GlcNAcT
7-8, GlcNAc-S-pNP, and
UDP-[3H]-GalNAc, was analyzed by lectin affinity
chromatography with Wisteria floribunda lectin (WFA),
immobilized on azlactone/bisacrylamide polymeric beads (3 M EmphazeTM, Pierce) (24). The product was also analyzed by
HPAEC-PAD (3). As a control, both WFA lectin chromatography and
HPAEC-PAD analysis were performed with reference GalNAc
1
4GlcNAc-S-pNP produced with L. stagnalis
albumen gland
4-GalNAcT (9).
A genomic clone, denoted 5, was isolated previously from
a
EMBL3A library of L. stagnalis, and was shown to
contain two short DNA sequences identical to
4-GlcNAcT cDNA
sequences (3). A rough genomic map of
5 was constructed by PCR and
Southern blot hybridization using specific
4-GlcNAcT cDNA
fragments as probes (Fig. 1).
5 was found to
encompass the complete coding sequence of the
4-GlcNAcT gene,
spanning 12.5 kb of DNA, that was divided into 10 exons (Fig. 1, Table
I). As probably part of the 5
-noncoding sequence is
lacking from the cDNA (3), we cannot exclude the presence in the
gene of one or more exons upstream of the denoted exon 1. Exon 10 was
found to encompass the complete 3
-noncoding region. All exon sequences
in the genomic clone were identical to those of the cDNA (3). Donor
and acceptor splice junction sequences (Table II) are in
agreement with consensus sequences reported (25).
|
|
The cDNA encoding 4-GlcNAcT
shows sequence identity with the mammalian
4-GalT cDNAs
identified (3, 5-7). A comparison of the protein-coding exons of the
L. stagnalis
4-GlcNAcT gene with those of the murine and
human
4-GalT genes (15, 16) is shown in Fig. 2. The
4-GalT gene was found to be divided into six exons, whereas the
4-GlcNAcT gene appeared to contain 10 exons. Exons 3, 4, 5, 6, and 9 of the
4-GlcNAcT gene were found to show similarity to exons 2-6 of
the
4-GalT gene, corresponding to the catalytic domain of the enzyme
(Fig. 2). This similarity was not only confined to a high degree of
sequence identity; intron/exon boundaries were also found at identical
positions within the gene.
The coding sequence of the 4-GlcNAcT gene is larger than that of the
4-GalT gene. These additional sequences appeared to be mainly
encoded by three exons (exons 7, 8, and 10), that were not present in
the
4-GalT gene. Exons 7 and 8 were found to encode a partial repeat
of the sequence of exon 6 (Table III). The sequence analysis of the intron/exon borders (Table II) shows that exons 6, 7, and 8 are symmetrical exons. Additionally, it was observed that the
acceptor splice junction sites of introns 6 and 7 as well as the donor
splice junction sites of intron 6, 7, and 8 and the adjacent exon
sequences are identical. These data strongly suggest that exons 7 and 8 of the
4-GlcNAcT gene arose by duplications (26), originating from
the downstream part of exon 6.
|
Deletion derivatives of the 4-GlcNAcT
cDNA were constructed that lacked the sequences corresponding to
exon 8 or to exons 7 and 8 (Fig. 3). Plasmids were
constructed as gene fusions to part of protein A (20, 27). pMC 135 encodes a soluble, native
4-GlcNAcT (protA-
4-GlcNAcT). pMC142 and
pMC166 are plasmids encoding deletion derivatives of pMC135, designated
protA-
4-GlcNAcT
7-8 and protA-
4-GlcNAcT
8, respectively.
These three plasmids were transiently expressed in COS cells.
Essentially equal amounts of hybrid protein per volume were secreted
into the medium (Fig. 4).
Enzyme Activity of the Chimeric Proteins
The enzyme
activities of the mutant hybrid proteins and the native
protA-4-GlcNAcT were assayed using similar amounts of enzyme (Fig.
4). As the native membrane-bound
4-GlcNAcT shows a low GalNAcT
activity (about 7% of the
4-GlcNAcT activity), the activity of the
enzymes was measured with both UDP-GlcNAc and UDP-GalNAc.
ProtA-
4-GlcNAcT
7-8 showed reproducibly an almost 2 times higher
GlcNAcT activity and a 4 times higher GalNAcT activity than the
parental protA-
4-GlcNAcT (Table IV). In contrast to the other chimeric proteins, protA-
4-GlcNAcT
8 appeared to be enzymatically inactive. To determine if the enzymes would show GalT
activity, the hybrid proteins were purified using IgG-agarose beads. In
this way, the recombinant enzymes were completely disposed of COS
cell-derived
4-GalT. The bead-associated chimeras did not show
detectable GalT activity, whereas they showed GlcNAcT and GalNAcT
activities similar to those for the concentrated media (results not
shown).
|
Acceptor specificity studies, using UDP-GlcNAc as sugar donor, showed
no significant differences in acceptor preference between the two
active hybrid enzymes at an acceptor concentration of 1 mM
(Table V; Ref. 4). The acceptor specificity of the
mutant chimeric protein using UDP-GalNAc as a sugar-donor appeared very similar to the preference of the enzyme when using UDP-GlcNAc. By
contrast, the prostate gland 4-GalNAcT is less specific for the
linkage type of the terminal
-linked GlcNAc, and its acceptor substrate requirement resembles that of the albumen gland
4-GalNAcT (9).
|
To explain
the differences in donor specificity in more detail, the
Km and V values for UDP-GlcNAc and
UDP-GalNAc were determined for both protA-4-GlcNAcT and
protA-
4-GlcNAcT
7-8. It appears that the increase in GlcNAcT
activity of protA-
4-GlcNAcT
7-8 is due to a 3-fold reduction in
Km for UDP-GlcNAc (Table VI), whereas
the increased GalNAcT activity of the mutant enzyme is mainly due to an
enhanced maximum velocity. These effects result in an elevated kinetic
efficiency for both types of transfer. The data suggest an involvement
of the region encoded by exons 7 and 8 in UDP-sugar donor binding.
However, it was found that both active chimera were inhibited to almost
the same extent by UDP (50% at 4-5 mM UDP, results not
shown).
|
The Km and V values of two acceptor substrates were estimated (Table VII). While the V values of both enzymes appeared to be of the same order for both compounds, the Km values were decreased in the mutant enzyme. Deletion of the two exons clearly elevates the kinetic efficiency with both acceptor substrates.
|
The
N-acetylgalactosaminylated product formed by incubating
protA-4-GlcNAcT
7-8, UDP-GalNAc, and GlcNAc-S-pNP was anticipated to be GalNAc
1
4GlcNAc-S-pNP. To confirm this, the product was compared with authentic GalNAc
1
4GlcNAc-S-pNP obtained by the action of L. stagnalis albumen gland
4-GalNAcT (9). Both
product and reference were subjected to lectin affinity chromatography with immobilized WFA, known to bind with high affinity to glycans containing terminal GalNAc in a
1
4-linkage (24, 28, 29). More
than 90% of both compounds bound to the immobilized WFA, and could be
eluted with 10 mM GalNAc. In addition, reference and
product eluted on HPAEC with the same retention time (data not shown).
These results indicate that
4-GlcNAcT
7-8 shows a
4-GalNAcT
activity.
Based on the sequence identity of the mammalian 4-GalTs and
L. stagnalis
4-GlcNAcT, these enzymes have been proposed
to be members of one gene family (3). The observed conservation of the
exon-intron organization of the genes encoding these enzymes strengthens this view. All exons of the
4-GalT gene show sequence identity to parts of the
4-GlcNAcT gene, which suggests that both
genes originated from an common ancestor, and diverged to genes
encoding enzymes with a different sugar donor specificity.
The most remarkable difference between the genes is found in an
insertion of two exons (exons 7 and 8) in the 4-GlcNAcT gene. These
exons were found to encode partial repeats of exon 6. The sequences of
these exons and those of the bordering introns strongly suggest that
exons 7 and 8 were generated by internal exon duplications, and
originate from exon 6. This is the first glycosyltransferase described
in which exon duplication seems to have taken place during evolution.
Generally, exon duplication is thought to play an important role in the
evolution of genes, and many complex genes have been described that
have evolved by internal exon duplication and subsequent modification
of the primordial genes (30, 31).
To study the effect of the exon duplications on enzyme catalysis, we
deleted the sequence (52 amino acids) corresponding to the two
additional exons from the cDNA sequence. The catalytic domain of
the mutant protein thus obtained, might resemble that of a putative
ancestor of the L. stagnalis 4-GlcNAcT. The deletion enhanced the kinetic efficiency of the enzyme for both the transfer of
GlcNAc and GalNAc, as well as for its acceptor substrates. The acceptor
substrate specificity, i.e. the preference for a terminal
6-linked GlcNAc, was not affected. So the mutant enzyme has an
enhanced catalytic potential, but also an increased sugar-donor promiscuity. Surprisingly, deletion of only the sequence encoded by
exon 8 resulted in a completely inactive enzyme. An explanation could
be that the enzyme is not properly folded with only one additional
repeat, whereas the presence of two additional copies of the repeated
sequence allows a correct folding.
From the results obtained here, it is difficult to deduce the selective
advantage that was obtained by the exon duplications for the biological
function of the 4-GlcNAcT. As can be inferred from the sequence,
changes have occurred in the repeats after the exon duplications, and
most likely also in other regions of the protein. It is possible,
however, that reduction of the capacity of the primordial
4-GlcNAcT
to transfer GalNAc might have been advantageous for the snail. The
increase in the specificity of the enzyme for UDP-GlcNAc would then
have been of more importance for the snail than the loss of catalytic
potency that coincided with the introduction of the additional
exons.
In 4-GalT the corresponding region (encoded by exon 5) has been
proposed to be involved in UDP-Gal binding (32-34). In the same region
a tetrapeptide (DKKN) has been found that is conserved between
4-GalT and
3-GalT, which is in support of this proposition (35).
In the human
4-GalT a second region (in exon 4) has been proposed to
be involved in UDP-Gal binding (36, 37). Interestingly, this latter
region shows a high degree of similarity between the
4-GalTs and the
4-GlcNAcT, whereas the more downstream region of
4-GalT (encoded
by exon 5) shows much less sequence identity with the corresponding
region in the
4-GlcNAcT (encoded by exons 6, 7, and 8). As the
mammalian
4-GalTs and the L. stagnalis
4-GlcNAcT use
UDP-Gal and UDP-GlcNAc, respectively, it is tempting to assume that the
domain that is most conserved between these enzymes, is involved in the
interaction with the UDP part of the nucleotide-sugar, while the more
downstream region is responsible for the specificity for donor Gal and
GlcNAc, respectively. The observation that both native
4-GlcNAcT and
the deletion mutant were inhibited to a similar extent by UDP,
suggesting that the UDP-binding domain is not affected by the deletion,
is in support of this supposition. The lower Km for
UDP-GlcNAc that was found for the mutant enzyme could be explained by a
higher affinity of this enzyme for the GlcNAc part of the
UDP-sugar.
The change in kinetic properties observed with the mutant enzyme
suggests that the region around the insertion in 4-GlcNAcT is not
only involved in interaction with the sugar-donor, but also in binding
of the acceptor substrate. Additionally, in
4-GalT a region (in exon
4) has been identified, that is involved in interaction of both donor
and acceptor substrates (36, 37). So in both enzymes the donor and
acceptor substrate binding domains seem to be structurally linked. This
is conceivable, as both substrates have to be close together for the
transfer reaction. In
4-GalT another binding domain for GlcNAc,
however, has been localized in exon 2/3 (34). This region shows a high
sequence identity with the corresponding region in
4-GlcNAcT, which
supports the suggestion that this sequence is involved in acceptor
binding.
We have shown here that the L. stagnalis 4-GlcNAcT can be
transformed to an enzyme that is capable of catalyzing the transfer of
GlcNAc and GalNAc with a similar maximum velocity. This suggests that
the
4-GalNAcT, which has been observed in L. stagnalis
tissues (Ref. 9 and this study), might be encoded by a structurally related enzyme belonging to the
4-GalT gene family. This is further supported by several studies that have shown a similarity between invertebrate
4-GalNAcTs and the mammalian
4-GalTs in acceptor specificity (9-12), and sometimes in responsiveness to
-lactalbumin (13). Furthermore,
4-GalT shows sugar donor promiscuity at high
concentrations of UDP-GalNAc (38), or in the presence of
-lactalbumin (24), resulting in the transfer of GalNAc.
Interestingly,
4-GlcNAcT is not sensitive to
-lactalbumin (4),
and this enzyme can not utilize UDP-Gal, but shows a relatively high
4-GalNAcT activity (7%). By mutagenesis, we have constructed an
enzyme with an even higher GalNAcT activity. A similar experiment has
been documented for the blood group A (
3-GalNAcT) and B (
3-GalT) enzymes (39). These enzymes also show some nucleotide sugar donor
promiscuity (40), but by construction of hybrids an enzyme with both
activities was obtained (41). Our observations suggest that, in
addition to the facilitation of the transfer of the desired sugar, the
prevention of sugar donor promiscuity might have been a driving force
in the evolution of enzymes of the
4-GalT gene family.
We thank J. Aino Andriessen, Casper Kee, and Carolien Koeleman for technical assistance in part of the experiments; Dr. Timo K. van den Berg for the gift of monoclonal antibody ED3; Dr. Bruce A. Macher for the gift of pPROTA vector; Dr. Ole Hindsgaul for the gift of oligosaccharide compound 4; and Dr. Richard D. Cummings for advice on lectin chromatography and the gift of WFA-EmphazeTM.