(Received for publication, July 7, 1995)
From the
Polysialic acid, or PSA, is a term used to refer to linear
homopolymers of (2,8)-sialic acid residues displayed at the
surface of some mammalian cells. PSA is typically linked to the neural
cell adhesion molecule N-CAM, where it can modulate the homotypic
adhesive properties of this polypeptide. PSA expression is
developmentally regulated, presumably through mechanisms involving
regulated expression of sialyltransferases involved in PSA
biosynthesis. Several different sialyltransferase sequences have been
implicated in PSA expression, although the precise roles of these
enzymes in this context remain unclear. One such sequence, termed STX,
maintains approximately 59% amino acid sequence identity with another
sialyltransferase (PST-1, from hamster; PST, human) that is known to
participate in PSA expression. While a murine STX fusion protein can
catalyze the synthesis of a single
(2,8)-sialic acid linkage in vitro, the ability of STX to participate in PSA expression in vivo has not been demonstrated. We show here that STX
transcripts are present in a PSA-positive, N-CAM-positive human small
cell carcinoma line (NCI-H69/F3), but are absent in a variant of this
line (NCI-H69/E2) selected to be PSA-negative and N-CAM-positive. To
functionally confirm this correlation, we have cloned a human cDNA
encoding the human STX sequence, and show, by transfection studies,
that human STX can restore PSA expression when expressed in the
PSA-negative, N-CAM-positive small cell carcinoma variant. We
furthermore show that STX can confer PSA expression when expressed in a
PSA-negative, N-CAM-positive murine cell line (NIH-3T3 cells), or when
expressed in PSA-negative, N-CAM-negative COS-7 cells. These
observations imply that STX, like PST-1/PST, can determine PSA
expression in vivo. When considered together with the
correlation between STX expression and PSA expression in vivo in the brain, these results suggest a regulatory role for STX in
PSA expression in the developing central nervous system and small cell
lung carcinoma.
Sialyltransferases represent a family of terminal
glycosyltransferases that catalyze the attachment of sialic acid to
carbohydrates of many glycoproteins and glycolipids(1) . Sialic
acids are key determinants of carbohydrate structures involved in a
variety of biological processes and are widely distributed on many cell
types (2, 3, 4) . Homopolymers of sialic
acids in (2,8) linkage (polysialic acid, PSA, (
)also
abbreviated as polySia) have a more restricted spatio-temporal tissue
distribution pattern than the more commonly found
(2,6)- and
(2,3)-linked sialic acid residues. For example, PSA is expressed
by neuronal tissues, in the heart and the developing kidney, and in
association with malignant transformation, such as in small cell lung
carcinoma (SCLC)(5, 6, 7, 8) .
Furthermore, PSA has been reported to be associated with only two
proteins, the neural cell adhesion molecule (N-CAM) and sodium channel
subunits(9, 10) . Changes in the amount of PSA
on N-CAM modulates the adhesive properties of N-CAM, and also affects
the cell surface properties of other molecules like some integrins,
N-cadherin, and
G4/NgCAM(11, 12, 13, 14) . These
effects are presumed to be due to the unusual physicochemical
properties of this very large, abundant, negatively charged, and linear
cell surface polyglycan(14) .
A requirement for N-CAM as an
acceptor molecule in PSA synthesis is implied from several
studies(15, 16) , although this has not been
demonstrated directly. Other studies suggest that PSA biosynthesis
involves the concerted activity of two or more specific
sialyltransferases(16, 17) . This includes a
requirement for one or more (2,3)-sialyltransferases to create
(2,3)-linked sialic acid moieties, that in turn are the presumed
acceptor substrate for subsequent addition of
(2,8)-linked sialic
acid moieties(18, 19) . It is possible that PSA
synthesis then proceeds through a two-step process involving the
addition of a single
(2,8)-linked sialic acid residue to the
(2,3)-linked sialic acid by one distinct
(2,8)-sialyltransferase (an ``initiase'' reaction),
followed by the addition of multiple
(2,8)-linked sialic acid
residues that yield PSA by a second distinct
(2,8)-sialyltransferase (a ``polymerase'' reaction).
This possibility is supported by in vitro experiments
indicating that at least three different
(2,8)-sialyltransferases
(ST8SiaI, GD3 synthase, (20, 21, 22) ;
ST8SiaII, STX, (23) ; ST8SiaIII, (24) ) can catalyze
the attachment of a single
(2,8)-linked sialic acid residue to
terminal
(2,3)-sialic acid linkages.
Alternatively, a single
(2,8)-sialyltransferase may operate to directly catalyze PSA
synthesis on a glycoconjugate template containing a terminal
(2,3)-linked sialic acid. This notion is supported by the recent
demonstration that expression of a single
(2,8)-sialyltransferase
gene, termed PST-1(25) , or PST(26) , is sufficient for
the expression of PSA in N-CAM-positive, PSA-negative mammalian cell
lines (CHO-2A10, NIH-3T3, and COS-hN-6, a COS cell line expressing with
human N-CAM-140 cDNA, (25) ; COS-1 cells and HeLa cells
transfected with an N-CAM expression vector, (26) ). In
principle, both such modes of PSA synthesis may exist, although this
remains to be confirmed, since it has not yet been possible to recreate
polymerization of
(2,8)-linked sialic acids in vitro.
The STX gene represents a member of the sialyltransferase gene
family whose developmentally regulated expression patterns correlate
well with PSA expression in certain
tissues(1, 24, 27) . A role for this sequence
in PSA expression remained circumstantial, however, since initial
efforts failed to demonstrate an enzymatic activity associated with the
(rat) STX polypeptide(27) . Subsequent efforts have
demonstrated that a recombinant (mouse) STX-protein A fusion protein
can catalyze the synthesis of a single (2,8)-sialic acid linkage in vitro(23) . Nonetheless, a definitive
demonstration that STX participates in PSA expression, in vitro or in vivo, has not been accomplished.
We show here that STX expression correlates with PSA expression in a PSA-positive human small cell lung carcinoma (SCLC) cell line (NCI-H69/F3)(8) , whereas STX transcripts are absent from a variant of this line selected to be PSA-negative (NCI-H69/E2)(8) . Transfection of the PSA-negative variant SCLC line with an STX expression vector restores PSA expression in that line, and in other PSA-negative cell lines. The observations imply that transcriptional regulation of STX can regulate PSA expression, suggest that STX shares overlapping enzymatic activity with PST-1/PST, and imply that determination of PSA expression by STX can be independent of N-CAM expression.
Recent molecular cloning work demonstrates that a
sialyltransferase sequence termed PST-1, or PST, (for
polysialyltransferase) can yield cell surface PSA expression when
expressed in PSA-negative cell lines(25, 26) .
Although these data implicate PST-1/PST in PSA expression, an in
vitro enzymatic correlate for these observations is not yet
available. Among all known sialyltransferases, PST-1/PST is most
similar to one termed STX (59% overall amino acid sequence identity;
hamster PST-1 or human PST versus rat STX). STX is expressed
primarily in the fetal and newborn brain, but not in the adult brain,
and thus correlates with the temporal sequence of PSA expression in
that tissue. These considerations suggest a role for STX in the
developmental regulation of PSA expression in the central nervous
system. However, efforts to assign an enzymatic activity to the rat STX
sequence have not met with success(27) . More recent work
indicates that, in vitro, mouse STX can utilize radiolabeled
CMP-sialic acid to incorporate radiolabeled sialic acid into
sialylated, N-linked glycoproteins(23) . Indirect
evidence suggests that (2,8)-sialic acid linkages were formed by
STX in these experiments, although there is currently no direct
evidence that STX participates in PSA biosynthesis or expression.
Figure 1:
Flow cytometry analyses of
PSA expression. A, SCLC cell lines NCI-H69/F3 and E2. Cell
lines NCI-H69/F3 and E2 were analyzed by flow cytometry, following
staining with the anti-PSA monoclonal antibody mAb 735 ( PSA, solidline), with a polyclonal rabbit
anti-human N-CAM antibody (
N-CAM, solidline), or with negative control antibodies (mouse IgG for
PSA; rabbit IgG for
N-CAM; dottedlines). B, mammalian cell lines transiently transfected with a vector
(pSTXFL) encoding human STX. PSA-negative cell lines (NCI-H69/E2,
NIH-3T3, COS-7) were transfected with a mammalian expression vector
encoding human STX (pSTXFL, solidlines), or with a
negative control vector (pCDNAI, dottedlines), as
described under ``Experimental Procedures.'' Transfected
cells were then stained with anti-PSA monoclonal antibody mAb 735 (
PSA), or with a negative control antibody, and were
subjected to flow cytometry analysis as described under
``Experimental Procedures.''
Northern blot analyses indicate that the
PSA-positive line NCI-H69/F3 is deficient in transcripts corresponding
to PST-1/PST (although PST-1 transcripts are observed in human heart; Fig. 2B), indicating that this gene does not
participate in PSA expression in these cells. Likewise, this cell line
is deficient in GD3 synthase and mST8Sia III transcripts (data not
shown). However, these analyses demonstrate that the STX transcript is
easily detectable in the PSA-positive line (Fig. 2A).
The STX transcript in the PSA-positive SCLC line (Fig. 2A) is similar in size (6.0 kb) to the human
(5.7 kb, human heart; data not shown and (1) ) and rat (5.5 kb, (27) ) STX transcripts, and corresponds roughly to broad range
(1.7-6.7 kb) of STX transcripts observed in the embryonic
mouse(24) . By contrast, STX transcripts are absent from the
PSA-negative variant SCLC line (Fig. 2A). These
observations suggest that PSA expression in this pair of cell lines is
controlled by transcriptional regulation of the STX gene.
Figure 2:
Northern blot analysis of the PSA-positive
and PSA-negative SCLC cell lines. A, Northern blot hybridized
with a human STX probe. The Northern blot was prepared using
poly(A) mRNA (2 µg/lane) isolated from the
PSA-positive SCLC cell line (PSA+, NCI-H69/F3), from the
PSA negative SCLC cell line (PSA-, NCI-H69/E2), and from
a mouse embryo at embryonic day 17 (E17). The blot was probed
with a segment of the human STX cDNA (``STX''), as described
under ``Experimental Procedures.'' B, Northern blot
hybridized with a hamster PST-1 probe. The blot shown in panelA was stripped of the STX probe (29) and
hybridized with a segment of the hamster PST-1 gene (PST-1), as described under ``Experimental
Procedures.'' Human heart (Heart) poly(A)
RNA served as a positive control(26) . C,
Northern blot hybridized with a human glyceraldehyde-3-phosphate
dehydrogenase probe (GAPDH). The blot shown in panelsA and B was stripped of the STX probe (29) and was hybridized with a segment of the human
glyceraldehyde-3-phosphate dehydrogenase gene, as described under
``Experimental Procedures.'' RNA molecular size standards are
indicated on the left (in
kilobases).
Figure 3: Human STX cDNA and predicted amino acid sequences. The DNA sequence of the coding region of the human STX is shown above the predicted amino acid sequence. The predicted signal anchor sequence is doublyunderlined and italicized. Potential asparagine-linked oligosaccharide attachment sites are underlined. Amino acids underlined with a dottedline correspond to consensus sequences identified previously for mammalian sialyltransferases (the ``L'' and ``S'' sialylmotifs; Refs. 1 and 20).
The bulk of PSA
expression in mammalian tissues and cell lines is thought to be
associated with N-CAM (9, 10, 36) and has
been directly demonstrated on the SCLC cell line
NCI-H69/F3(8) . These observations have been taken to mean that
effective PSA synthesis and expression requires N-CAM, although PSA has
also been detected in association with sodium channel
subunits(10) . To directly determine if STX-directed PSA
expression involves a requirement for N-CAM, STX was expressed in
N-CAM-negative (data not shown) COS-7 cells, and the transfectants were
assayed for PSA expression. More than 25% of the STX-transfected COS-7
cells stained with the anti-PSA monoclonal antibody mAb 735, and at
levels roughly comparable to the N-CAM-positive STX transfectants.
Control COS-7 transfectants remained PSA-negative. Considered together,
these results indicate that STX can also determined PSA expression in
the absence of N-CAM expression.
The nature of the glycoconjugates
that display STX-determined PSA on the cell lines we have used will
require detailed structural analysis. Our experiments also do not allow
us to know if STX participates in PSA expression as an
``initiase'' only (adds sialic acid in (2,8) linkage to
an
(2,3)-linked sialic acid precursor), or as a
``polymerase'' only (adds sialic acid in
(2,8) linkage
to an
(2,8)-linked sialic acid precursor), or if both of these
reactions are catalyzed by STX. A full biochemical resolution of this
question, as it relates to STX, and to the other
(2,8)-sialyltransferases implicated in PSA expression, will
require the development of an in vitro assay for PSA
synthesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33551[GenBank].