From the
Two cDNAs encode rat intestinal alkaline phosphatases having
completely different carboxyl-terminal peptides; one is hydrophobic and
fulfills the consensus requirements for glycan phosphatidylinositol
linkage, and the other is neither hydrophobic nor hydrophilic, but
contains a small amino acid domain (-NSASS-) just distal to a region of
17 threonine residues. Constructs were created using 80% of the
amino-terminal portion of one alkaline phosphatase and the
carboxyl-terminal portions of each of the isoforms. Both of the
carboxyl-terminal peptides supported glycan phosphatidylinositol
linkage as demonstrated by the following criteria: 1) plasma membrane
targeting in transfected COS-l cells, 2) release of transfected
alkaline phosphatase by phosphatidylinositol-specific phospholipase C,
3) appearance of the trypanosome variable glycoprotein cross-reacting
determinant after phospholipase C treatment, 4) ethanolamine
incorporation into newly synthesized enzyme, 5) loss of phospholipase C
release after mutation of the
Over 95% of rat intestinal alkaline phosphatase
(IAP)
Cell-free translation of rat intestinal RNA has yielded two
independently regulated IAP isoforms (62 and 65 kDa)
(4) that
show different sugar content of glycosyl side chains
(5) . This
suggested that two IAP isoforms were encoded in individual and distinct
RNAs. The first cDNA isolated encoding a rat IAP (IAP-I) had a
predicted structure of the carboxyl-terminal peptide that differed
markedly from IAP sequences predicted in other species
(6) .
Evidence from studies using a portion of the cDNA sequence at the 3`
end of the coding region, as well as the full-length cDNA as probes,
suggested that the 2.7- and 3.0-kb mRNAs encoding rat IAP each
represented a different isoform
(7) . Two different groups
reported a second rat IAP cDNA (IAP-II), which differs most strikingly
from the predicted structure of IAP-I in the carboxyl-terminal
region
(8, 9) . Thus, unlike the mouse and human, the rat
intestine expresses two separate IAP isoforms, most likely derived from
two separate genes.
Rat IAP-I appeared bound to membranes after
transfection into COS-l cells via a glycan phosphatidylinositol (GPI)
linkage
(6) . The cDNA encoding this protein contains a
carboxyl-terminal hydrophobic amino acid sequence preceded by a small
amino acid domain (-NSA-) located 28 residues from the end of the
predicted amino acid sequence. These features are consistent with the
requirements suggested for GPI linkage (10). On the other hand, the
predicted carboxyl-terminal sequence of rat IAP-II differed
significantly from that of rat IAP-I and would not appear to contain
the consensus requirements for GPI linkage. The 24 amino acids at the
carboxyl terminus were predicted to be either neutral or hydrophilic
(8) and were preceded by a distinctive segment of 17 threonine
residues. These findings suggested that rat IAP-II might be attached to
membranes via a non-GPI linkage or else secreted as a soluble protein.
The present experiments investigated the possibility that rat IAP-II
is not linked by a GPI mechanism. Experiments were conducted to examine
the anchoring of both IAP-I and IAP-II to the cell plasma membrane and
to secreted SLPs. A series of DNA constructs were used with altered
carboxyl-terminal sequences. The data show that both IAP-I and IAP-II
are membrane-bound via a GPI linkage, and that both are also included
after transfection in the secreted SLP produced by Caco-2 cells.
Because IAP-II and
the chimeric IAP contained no hydrophobic carboxyl-terminal
peptide
(8) , two further assays were used to document the
membrane-bound nature of the expressed product of the chimeric DNA. The
peak of IAP activity on NaBr gradient was at d = 1.08
(Fig. 2), as expected for SLP, not at d = 1.06 as
seen with solubilized IAP
(3) . When concentrated media from
chimeric transfections were applied to a Sepharose 6B column, the peak
of IAP activity eluted with the void volume (Fig. 3), well before
the R
In the chimeric IAP the
putative GPI linkage site is -NSA-, 4-6 residues beyond the
oligothreonine sequence and 19-21 residues from the predicted end
of the peptide. An Asn
Direct demonstration of the
GPI linkage for the chimeric IAP was obtained by documenting the
appearance of the trypanosome variable surface glycoprotein
cross-reacting determinant (CRD) after PI-PLC treatment and by
incorporation of ethanolamine into the newly synthesized product of the
chimeric IAP DNA. Fig. 5demonstrates that anti-CRD antibody,
which recognizes the inositol phosphate moiety exposed by PI-PLC
hydrolysis, also recognizes the product of both IAP-I and the chimeric
IAP DNA expressed in COS-l cells. The apparent molecular mass of IAP-I
was 65 kDa, as previously reported
(6) . The chimeric IAP product
was larger (about 90 kDa), despite the fact that most of the coding
region of the two constructs was identical. Media from nontransfected
COS-1 cells showed no reaction with anti-CRD antiserum. Transfected
cells were also incubated with [
The deduced
sequence of IAP-I
(8) fits the current requirements for GPI
linkage. It contains a 22-amino acid hydrophobic carboxyl-terminal
peptide, a hinge region of 5 residues, including two charged ones
(-NN-), and a 3-amino acid sequence with high probability for linkage
(-NSA-) at positions -28-30. Another possibility is the
sequence one residue shifted (-SAI-), although the probability of
isoleucine being active in the
Most evidence to date suggests that a hydrophobic signal
peptide is necessary for GPI linkage
(10, 15) . In fact,
amino-terminal signal peptides have been substituted for the native
carboxyl-terminal sequence with retention of GPI linkage
(16) .
There is one example of presumed GPI linkage without a hydrophobic
carboxyl-terminal signal peptide. A truncated version of platelet
derived growth factor receptor was produced that removed the
transmembrane and cytoplasmic domains and terminated with the predicted
carboxyl-terminal sequence -VTSGHCHEERVDRHDGE
(17) .
Surprisingly, this construct was membrane-bound and was released by
PI-PLC treatment. This protein was not naturally occurring, as is
IAP-II, and the truncated receptor was not normally targeted, with only
20% of the protein found on the cell surface, the rest being
intracellular. The product of IAP-II cDNA transfection was located as
expected for a GPI-linked protein at the cell surface, as demonstrated
by immunocytochemistry and by PI-PLC release.
Although a hydrophobic
carboxyl-terminal peptide is not present in IAP-II, there is a possible
linkage site (-NSA-), similar to that found in IAP-I, but located in
IAP-II at positions -17-21. Another possible linkage site
(-NST-) is located just at the amino-terminal side of the
poly-threonine sequence. Threonine in the
The truncated IAP isoform removed the putative GPI linkage site at
positions -19-21 in IAP-II, while retaining the
poly-threonine sequence and the other putative GPI linkage site, -NST-
(Fig. 1). The product of this transfected cDNA was not
membrane-bound and was secreted in a form not associated with
membranes. Thus, the most likely predicted site for GPI linkage in
IAP-II is -NSA-. The experiments utilizing mutations in the -NSASS-
region further support this conclusion. The double mutation,
Ala
The unique feature of the
IAP-II sequence is the region of 17 threonine residues. Although there
is no direct evidence that these form the carboxyl terminus of the
protein, the results of transfections with the truncated IAP and
mutated chimeric IAP suggest that they do, because the putative GPI
linkage site (-NSA-) would be carboxyl-terminal to the threonine
sequence. These threonines could be the site for O-linked
glycosylation or some other modification. Such modification could
result in the increased apparent size of IAP-II compared with IAP-I (93
versus 69 kDa, Fig. 6). N-Linked glycosylation
seems an unlikely single explanation, because the products of cell-free
translation of rat duodenal mRNA are 62 and 65 kDa and after incubation
with microsomal membranes 69 and 65 kDa, respectively
(4) .
O-Linked glycosylation or other modification of
carboxyl-terminal threonine(s) is possible, but evidence for or against
such an explanation is not available.
It is clear, however, that two
IAPs are found in the rat intestinal mucosa, as detected either by
purification
(5) or Western blotting (13). By studying the
appearance of IAP isoforms during postnatal development or after
precocious induction of development by cortisone and thyroxine, the
appearance of the 69-kDa IAP appears to be directed by the 2.7-kb mRNA
that encodes IAP-I, whereas the 93-kDa IAP is directed by the 3.0-kb
mRNA that encodes IAP-II
(13) . The present data support the
assignment of the 69- and 93-kDa IAP isomers to the IAP-I and IAP-II,
respectively.
It is difficult to believe that IAP-II is a unique
naturally occurring protein, i.e. the only one which is
GPI-linked yet lacking a hydrophobic carboxyl-terminal signal peptide.
It seems more likely that the requirement for GPI attachment will be
modified to include a variety of sequences which remain to be
elucidated. For the present, the carboxyl-terminal sequence of IAP-II
provides a unique naturally occurring model to be used for targeting
and for stimulation of SLP production.
Results
are expressed as the mean ± S.E. of three to seven experiments.
and
+ 2 positions in
the putative linkage site, -NSA-, and 6) evidence of surface membrane
localization by immunofluorescence using antibody against rat
intestinal alkaline phosphatase. These data demonstrate that a
predicted hydrophobic carboxyl-terminal sequence is not essential for
glycan phosphatidylinositol linkage. Moreover, because both isomers are
membrane-bound, the origin of soluble enzyme in the serum is likely to
arise from the action of serum phosphatidylinositol-specific
phospholipase D.
(
)
is localized to the apical brush border
membrane, yet this same enzyme contributes >70% of the total serum
alkaline phosphatase activity after fat feeding
(1) . This
response to fat feeding results from an increase in enzyme protein
concentration, not in activity
(2) . Because alkaline phosphatase
is a membrane-bound enzyme, a separate secretory pathway or a soluble
form of the enzyme, or both, would be needed to explain IAP in the
serum. We have found such a possible pathway for a membrane-bound IAP
via its association with a secreted phospholipid-rich membrane with
surfactant properties, the surfactant-like particle (SLP). This SLP is
characterized by enrichment for IAP, over 30 times greater than the
brush border content per mg of protein
(3) . In addition,
evidence for two separate IAPs in rat intestinal mucosa was found,
raising the question whether one isoenzyme was uniquely directed to the
SLP for secretion or was secreted by a different mechanism.
IAP cDNA Constructs
Rat IAP-I was ligated into
the BamHI site of the mammalian transfection vector pSG5
(Pharmacia Biotech Inc.)
(11) . All other modifications were made
from this construct (Fig. 1). (a) For chimeric IAP, the
3` end of the IAP-I insert was removed between NarI (bp 1532)
and BglII in the multiple cloning region of pSG5. A similar
digest (NarI-EcoRI) was made using the IAP-II DNA
which had been produced by PCR and subcloned into the TA cloning vector
(Invitrogen, San Diego, CA). This portion of IAP-II includes the most
3` 170 bp of the coding region as well as 1000 bp of the
untranslated region. The chimeric DNA produced by ligating this
NarI
EcoRI fragment into the restricted
pSG5/IAP-I vector encodes for the first 1531 bp of IAP-I and the final
170 bp of IAP-II. The protein resulting from translation of this DNA
should have both alkaline phosphatase activity from IAP-I and 57 amino
acids supporting the IAP-II carboxyl-terminal signal. (b) For
truncated IAP, the chimeric IAP DNA was digested with AvaI and
NheI (Fig. 1). This digestion removes the nucleotides
encoding the carboxyl-terminal 24 amino acids, but leaves intact the
unique stretch of 17 threonine residues found in IAP-II, as well as
350 bp of the 3`-untranslated region, including the poly(A) tail.
The single-point mutations were constructed by PCR using the chimeric
DNA as template and 5`-oligomers containing the desired base changes as
well as the AvaI restriction sequence at bp 1633. The
3`-oligomer was derived from a sequence in the untranslated region and
included the NheI restriction site at bp 2243. The chimeric
DNA and the PCR products were digested with AvaI and
NheI, and the PCR fragments were subsequently ligated into the
chimeric DNA. All constructs were verified by restriction digests and
DNA sequencing.
Figure 1:
The
carboxyl termini of the rat intestinal alkaline phosphatase constructs
indicating changes and mutations described under ``Materials and
Methods.''
Cell Culture and Transfection
Caco-2 cells
(courtesy of Dr. Jeffrey Field, University of Iowa) were used between
passages 40 and 45. The cells were grown in 100-mm diameter plastic
dishes as described previously
(11) . COS-1 cells (courtesy of
Dr. John Brady, National Cancer Institute, NIH) were grown in DMEM with
the same antibiotics used for Caco-2 cells. COS-1 cells were used
between passages 36 and 57. Transfections were performed either with
the calcium phosphate precipitation method (Caco-2 cells)
(11) or by electroporation (COS-1 cells). Optimal transfection
conditions for electroporation were 1000 µF, 300 V at
resistance, using the Invitrogen Electroporator (Invitrogen Corp.).
Cells were electroporated in 750 µl in 0.4-cm cuvettes. Control
transfections contained either pSG5 alone or no added DNA.
Treatment with Phosphatidylinositol-specific
Phospholipase C (PI-PLC)
PI-PLC prepared from Staphylococcus
aureus culture medium was generously provided by Dr. Martin G. Low
(Columbia University, New York, NY). COS-1 cells were washed three
times with serum-free DMEM and then incubated for 60 min at 37 °
with 3 ml of serum-free DMEM. The medium was removed, centrifuged at
600 g to remove cellular debris, and stored at
-20 °C. The cells were refed with 3 ml of serum-free DMEM
containing 60-80 milliunits/ml PI-PLC. Following a 60-min
incubation the medium was removed, centrifuged, and stored at -20
°C. Alkaline phosphatase activity was measured with 10 mMp-nitrophenyl phosphate as substrate. One unit of activity
equals the amount of enzyme needed to hydrolyze 1 mmol of substrate per
min at 37 °C. After assay for enzyme activity, the media from
replicates of the same transfection were pooled, concentrated with an
Amicon YM-10 membrane, and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Western blots were
performed as described previously
(12) , using an antibody to the
trypanosome-variable surface glycoprotein cross-reacting determinant
(CRD), kindly supplied by Dr. John Englund (Johns Hopkins University,
Baltimore, MD). The ECL detection system with linked horseradish
peroxidase (Amersham Corp.) was used for visualization of
antigen-antibody complexes. Media from nontransfected cells (containing
no rat IAP) was used as a control.
Ethanolamine Labeling Studies
Cells were incubated
overnight (16-20 h) in DMEM supplemented with 1% fetal bovine
serum and 100 µCi/ml of
[1-
H]ethan-1-ol-2-amine HCl, 15 Ci/mmol (Amersham
Corp.). Following the labeling period cells were scraped into 1 ml of
immunoprecipitation buffer to which 15 µl of antiserum raised
against native rat IAP were added
(11) . Following denaturing
polyacrylamide gel electrophoresis, the gels were treated with ENHANCE
(DuPont NEN) and exposed to X-Omat film (Eastman Kodak Co.) for
20-24 h. Apparent protein size was determined by comparing the
protein of interest to a set of molecular weight markers (Bio-Rad).
Assays for Membrane-bound Status of IAP
Isoenzymes
Three additional methods were used to document
whether the IAP isoforms were membrane-bound. Phase partitioning with
Triton X-114 and separation of free from membrane-bound IAP by NaBr
density gradient centrifugation have been described
previously
(11) . Chromatography with Sepharose 6B (Pharmacia) to
separate the intact SLP from solubilized IAP was performed as reported
earlier
(12) .
Fluorescence Microscopy
COS-1 cells were
transfected and grown in Chamber Slide culture chambers (Nunc Inc.,
Naperville, IL). Control and transfected cells were washed three times
with phosphate-buffered saline (pH 7.3) containing 0.1 mM
CaCl and 1 mM MgCl
(PBS-CM), followed
by a 15 min incubation at room temperature with 2% paraformaldehyde.
The cells were again washed three times with PBS-CM and then incubated
for 10 min at room temperature with 50 mM NH
Cl in
PBS to quench free aldehyde groups. After two washes with PBS-CM plus
0.2% gelatin (PBS-CMG), the cells were exposed to rat IAP antibody
(diluted 1:100 in PBS-CMG) for 30 min at room temperature. The cells
were next washed four times with PBS-CMG for 5 min each and incubated
for 30 min at room temperature with fluorescein
isothiocyanate-conjugated second antibody (Sigma) diluted 1:10 in
PBS-CMG. Cells were rinsed four times, 5 min each, with PBS-CMG and two
times for 5 min with PBS alone. Slides were mounted with 50% glycerol
in PBS, sealed with nail varnish, and stored at 4 °C in the dark
until examined. Slides were examined using a Nikon fluorescent
microscope.
RESULTS
Secretion of Membrane-bound Alkaline Phosphatase from
Caco-2 Cells
To determine whether both IAPs were membrane-bound
or whether IAP-II might be secreted in soluble form, the DNA constructs
encoding IAP-I and the chimeric IAP were transfected into Caco-2 cells.
These cells were chosen because alkaline phosphatase secretion
resulting from transfection with IAP-I DNA is mediated by a secreted
membrane, the SLP
(11) . Transfection with IAP-I DNA resulted in
a 29-fold increase in medium alkaline phosphatase activity
(). When chimeric DNA was used, the increase in activity
was approximately 220-fold. Enzyme activity alone, however, cannot
distinguish between soluble and SLP-bound IAP. The secreted product of
IAP-I was found mostly in the detergent-rich fraction of the Triton
X-114 partition assay (); the same result was found for the
secreted product of chimeric DNA transfection.
of soluble IAP. Identical results
were seen using media from IAP-I cDNA transfection (data not shown).
Thus, in the Caco-2 cell system, transfection with DNA encoding either
IAP-I or the chimeric IAP produced a secreted IAP which appeared to be
membrane-bound. We next examined the intracellular targeting of both
IAP isoenzymes in a system which did not produce a secreted form of
IAP.
Figure 2:
NaBr gradient fractionation of media from
Caco-2 cells transfected with the chimeric IAP construct. Media were
collected 72 h after transfection and prepared for the gradient as
described under ``Materials and Methods.'' One-ml fractions
were collected and assayed for alkaline phosphatase
activity.
Figure 3:
Sepharose 6B column chromatography of
media from Caco-2 cells transfected with the chimeric IAP construct.
Media from transfected cells were collected 72 h after transfection and
prepared as described under ``Materials and Methods.'' A 37
1.8-cm column was eluted with Tris-HCl buffer, pH 7.8. Void
volume fractions were numbers
8-12.
Transfection of IAP Isoforms in COS-1 Cells
COS-1
cells have low cellular levels of alkaline phosphatase activity and do
not secrete the enzyme under basal conditions
(6) . Thus, any
media IAP activity would result either from secreted protein or from
the action of serum proteases or lipases on membrane-bound IAP. COS-1
cells were initially transfected with DNA encoding IAP-I, the chimeric
IAP, the truncated IAP, and the chimeric IAP in which the amino acids
at position 533 and 535 had been mutated to prolines. Cells and media
were examined for alkaline phosphatase activity 72 h later
(). Cellular alkaline phosphatase activity was increased
143-, 122-, 97-, and 37-fold when transfected with DNA encoding IAP-I,
chimeric IAP, truncated IAP, and mutated chimeric IAP, respectively.
The four constructs produced increases in alkaline phosphatase activity
in the medium by 41-, 104-, 840-, and 189-fold. When expressed as a
percent of total activity (cell + medium), the cell-associated
activity was 78, 44, 7, and 13%, respectively (). These
data suggested that the carboxyl-terminal portion of both IAP-I and
IAP-II (chimeric IAP) were directed to membranes, whereas removal of
the carboxyl-terminal peptide or mutation of the probable GPI-linkage
site produced a soluble and secreted protein.
Localization of IAP Activity by
Immunocytochemistry
Immunofluorescence using anti-IAP antibody
and fluorescein isothiocyanate-labeled second antibody localized the
protein products of the transfected cDNAs. Fig. 4demonstrates
that the proteins expressed after transfection of cDNA encoding IAP-I
and the chimeric IAP were found predominantly on the external plasma
membrane, whereas control cells and cells transfected with the
truncated IAP were unlabeled.
Figure 4:
Immunofluorescent labeling of
nonpermeabilized COS-1 cells transfected with the following cDNAs:
A, control; B, IAP-I; C, chimeric construct;
D, truncated construct. Cells were fixed and incubated with
anti-IAP-I antibody as described under ``Materials and
Methods.''
Demonstration of GPI Linkage for IAP-I and
IAP-II
IAP-I has been demonstrated to be membrane-bound by a GPI
linkage
(6) and was used as a positive control for the
experiments that examined the possibility that the chimeric IAP might
also be anchored by a similar mechanism. COS-1 cells were transfected
with cDNA encoding IAP-I, the chimeric IAP, the truncated IAP, or a
series of mutated chimeric IAPs (see I). After 72 h cells
were incubated for 1 h either with or without PI-PLC, and the amount of
alkaline phosphatase activity released into the medium was assessed
(I). PI-PLC led to a 37-38-fold increase in medium
activity for both IAP-I and chimeric IAP-transfected cells. In
contrast, medium alkaline phosphatase activity was unchanged following
PI-PLC treatment of cells transfected with either the truncated IAP DNA
or the proline substituted chimeric IAP DNA.
Thr change in the putative
(attachment) site nearly eliminated IAP linkage that was sensitive to
PI-PLC, as did the mutation Ala
Pro in the
+ 2 site.
Mutation to Thr in the
+ 1,
+ 2,
+
3, or
+ 4 sites did not much affect PI-PLC sensitive
linkage. Threonine is known to support GPI linkage, although less
strongly than some other amino acid residues, whereas proline does not
support GPI linkage at all
(10) .
H]ethanolamine to
determine incorporation into the GPI moiety. Fig. 6shows that in
cells transfected with chimeric IAP DNA a single band of radioactive
immunoprecipitated IAP was seen, with an apparent molecular mass of 93
kDa; IAP resulting from transfection with IAP-I cDNA had an molecular
mass of 69 kDa. These data support previous observations of two
different IAP isoforms in rat intestinal tissue, in which the product
of the IAP-I gene corresponded with the 68-kDa isoform, and that of
IAP-II with the 90-kDa isoform
(4, 13) .
Figure 5:
Expression of the trypanosome-variable
cross-reacting determinant after PI-PLC treatment. COS-1 cells were
transfected with the following constructs: no cDNA (-); IAP-I
(I); chimeric construct with the IAP-II carboxyl terminus
(II); the truncated construct (Tr). Cells were
treated witih PI-PLC 72 h after transfection. Western blots on the
concentrated media were performed as described under ``Materials
and Methods.''
Figure 6:
Immunoprecipitation of
[H]ethanolamine-labeled COS-1 cells. Cells were
transfected with either IAP-I (I) or the chimeric construct
containing the IAP-II carboxyl terminus (II). Cell labeling
and immunoprecipitation were carried out as described under
``Materials and Methods.''
DISCUSSION
The data in this report demonstrate that IAP-II, like IAP-I,
is bound to membranes via a GPI anchor. The evidence for such a
conclusion is as follows. The products resulting from transfection with
cDNAs encoding these two proteins are bound to COS-1 cell membranes and
released from the cells by PI-PLC treatment, are not released by PI-PLC
after mutation of the putative -NSA- GPI linkage site, demonstrate
reactivity with anti-CRD antiserum after PI-PLC treatment, and
incorporate ethanolamine into the immunoprecipitated protein. Moreover,
when transfected into Caco-2 cells that secrete IAP attached to a
membranous SLP, both IAP isoforms are secreted in a form that
segregates with the detergent-rich phase of the Triton X-114 separation
system, consistent with their attachment to the SLP.
+ 2 position is only 10%
that of alanine
(14) . The predicted amino acid sequence deduced
from the IAP-II cDNA, however, should not support a GPI-linked
mechanism, if the same requirements are present. The carboxyl-terminal
peptide of IAP-II and of the chimeric IAP lacks the apparently
necessary hydrophobic span of 8-20 amino acids
(10) . As a
result, no hinge region can be identified, although a proline residue,
often found in hinge regions, is present at positions -10 and
-14.
+ 2 position
produces only low GPI-linkage activity
(14) , but at least one
natural protein, decay accelerating factor, has a threonine in this
position
(14) . Although this -NST- sequence in IAP-II lines up
nearly exactly in paired amino acid analysis with the putative -NSA-
sequence that is the most likely linkage site in IAP-I, the -NSA- site
in IAP-II seems likely to be utilized based on the mutational studies.
Pro/Ser
Pro, resulted in
a secreted protein. Proline was chosen for this substitution based on
studies
(14) , which found that proline was unable to support GPI
attachment when present in the
,
+ 1, or
+ 2 positions. Single amino acid mutations in the region of amino
acids 532-535 (-SASS-) did not alter the linkage mechanism of the
chimeric protein, indicating that none of these amino acids is the
attachment site. Substitution of amino acid 531 (Asn to Thr), however,
sharply reduced the amount of enzyme released by PI-PLC treatment, thus
identifying this asparagine as the most likely candidate for GPI anchor
attachment. The mutation of asparagine 531 to threonine did not
completely eliminate IAP release by PI-PLC, as did the truncated or
double-mutated chimeric IAP. The reason for this is not entirely clear,
although mutating only one amino acid in this region may shift the GPI
reading frame and permit attachment of the anchor to another, less
desirable, amino acid in this region.
Table:
Effect of transfection of alkaline phosphatase
cDNAs on enzyme secretion from Caco-2 cell
Table: Effect of transfection of alkaline
phosphatase cDNAs on enzyme localization in COS-1 cells
Table:
Effect of PI-PLC treatment on transfected
COS-1 cell alkaline phosphatase activity
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.