From the Glycobiology Program, Cancer Research
Center, The Burnham Institute, La Jolla, California 92037, the
§ Department of Chemistry and Biochemistry, San Francisco
State University, San Francisco, California 94132, and
¶ INSERM Unité 260 Faculté de Médecine, 27 Blvd.
J. Moulin, 13385 Marseille Cedex 5, France
Received for publication, January 22, 2001, and in revised form, February 7, 2001
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ABSTRACT |
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NCAM polysialylation plays a critical role in
neuronal development and regeneration. Polysialylation of the neural
cell adhesion molecule (NCAM) is catalyzed by two
polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST),
which contain sialylmotifs L and S conserved in all members of the
sialyltransferases. The members of the ST8Sia gene family, including
ST8Sia II and ST8Sia IV are unique in having three cysteines in
sialylmotif L, one cysteine in sialylmotif S, and one cysteine at the
COOH terminus. However, structural information, including how disulfide
bonds are formed, has not been determined for any of the
sialyltransferases. To obtain insight into the structure/function of
ST8Sia IV, we expressed human ST8Sia IV in insect cells,
Trichoplusia ni, and found that the enzyme produced in the
insect cells catalyzes NCAM polysialylation, although it cannot
polysialylate itself ("autopolysialylation"). We also found that
ST8Sia IV does not form a dimer through disulfide bonds. By using the
same enzyme preparation and performing mass spectrometric analysis, we
found that the first cysteine in sialylmotif L and the cysteine in
sialylmotif S form a disulfide bridge, whereas the second cysteine in
sialylmotif L and the cysteine at the COOH terminus form a second
disulfide bridge. Site-directed mutagenesis demonstrated that mutation
at cysteine residues involved in the disulfide bridges completely
inactivated the enzyme. Moreover, changes in the position of the
COOH-terminal cysteine abolished its activity. By contrast, the
addition of green fluorescence protein at the COOH terminus of ST8Sia
IV did not render the enzyme inactive. These results combined indicate
that the sterical structure formed by intramolecular disulfide bonds,
which bring the sialylmotifs and the COOH terminus within close
proximity, is critical for the catalytic activity of ST8Sia IV.
In the development of the nervous system, various
cell-type-specific carbohydrates presented by glycoproteins,
proteoglycans, and glycolipids function in signal transduction, neurite
outgrowth, neuronal plasticity, and synapse formation (1, 2). Among these carbohydrates, polysialic acid is a linear homopolymer of Polysialic acid is also detected in neuroblastoma (11), Wilms' tumor
(12), and lung carcinomas (13, 14). In both small cell and non-small
cell lung carcinomas, the expression of polysialic acid is associated
with tumor progression, in particular with tumor metastasis (13, 14).
In addition, capsules of certain pathogenic bacteria that cause
meningitis bear polysialic acid (15). These combined results suggest
that polysialic acid facilitates cell migration and allows pathogens to
avoid attack by the immune system in vivo. To develop
therapeutic agents for the treatment of these diseases and determine
the roles of polysialic acid in neural development, it is important to
understand the mechanisms of polysialic acid synthesis.
To date, two polysialyltransferases, ST8Sia IV (PST) and ST8Sia
II (STX), have been cloned and shown to synthesize polysialic acid on NCAM (16-20). These enzymes catalyze transfer of multiple Polysialyltransferases belong to the vertebrate sialyltransferase
gene family that includes
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,8-linked sialic acid, primarily attached to the neural cell adhesion molecule (NCAM)1
(3-5). Polysialylated NCAM is abundant in embryonic brain, whereas most NCAM in adult brain does not contain polysialic acid. However, polysialylated NCAM is continuously present in hippocampus and the
olfactory bulb, where neuronal regeneration persists in the adult (6).
Studies using endoneuraminidase showed that cell migration and synaptic
plasticity are dependent on the presence of polysialic acid (7, 8). In
contrast with vertebrate NCAM, NCAM homologues in Aplysia
and Drosophila, apCAM and FasII, respectively, are not
polysialylated. In these species, their activity in neuronal regeneration and axon guidance is modulated by removal of the protein
from the cell surface by endocytosis (9) or expression of an
anti-adhesive protein (10). These results indicate that polysialic acid
evolved in vertebrates and plays critical roles in formation and
remodeling of the nervous system as a common and unique regulator of
the adhesive property of NCAM in vertebrates.
2,8-linked sialic acid residues to an acceptor containing a
NeuNAc
2
3Gal
1
4GlcNAc
R structure without participation of
other enzymes (21-24), and the presence of polysialic acid is always
associated with expression of ST8Sia II and/or IV (25, 26). The amino
acid sequences of these mammalian polysialyltransferases are not
related to bacterial polysialic acid synthase (27). Recent studies
demonstrate that inactivation of ST8Sia IV in mice through homologous
recombination leads to impaired long term potentiation and long term
depression in Schaffer collateral-CA1 synapses of the adult hippocampus
(28). Loss of polysialic acid in the above mutant mice was incomplete, because ST8Sia II compensates for the loss of ST8Sia IV. These results
suggest that ST8Sia IV and ST8Sia II are the key enzymes that control
the expression of polysialic acid in vivo.
2,3-,
2,6-, and
2,8-sialyltransferases. Sialyltransferases have a type II
membrane protein topology as do almost all Golgi-associated
glycosyltransferases cloned so far. As generally seen for these
enzymes, a short cytoplasmic segment of ST8Sia II and IV is connected
to a transmembrane domain followed by a large intraluminal domain
consisting of a stem region and catalytic domain. Analysis of the amino
acid sequences of various sialyltransferases shows two weak but
discernible homologous regions in their catalytic domains, called
sialylmotifs L and S (29). It has been demonstrated by analysis of
ST6Gal I that sialylmotif L is involved in the binding to the donor
substrate, CMP-NeuNAc, whereas sialylmotif S participates in the
binding to both donor and acceptor substrates (30, 31). The members of
2,8-sialyltransferases (ST8Sia) are characteristic in having one
conserved cysteine at the COOH-terminal end, three cysteine residues in
the sialylmotif L, and one in the sialylmotif S (Fig. 1). On the other hand, all of the other
sialyltransferases contain only one conserved cysteine residue in each
sialylmotifs L and S. Except for ST6Gal I (32), none of the other
sialyltransferases contains a cysteine at the COOH terminus. Disulfide
bond structures have not been determined for any members of
sialyltransferases, and it is not known how sialylmotifs L and S
work together.
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Fig. 1.
Comparison of the amino acid sequences of
ST8Sia IV, ST8Sia II, ST8Sia III, ST8Sia V, ST8Sia I, and ST3Gal
I. N-Glycosylation sites are shown by the symbol
and numbered for ST8Sia IV. The transmembrane domain (TM),
stem region (stem), and sialylmotifs L (L) and S
(S) are indicated. The total number of amino acid residues
is shown. Cysteine residues are indicated by the circled
"c," and the conserved cysteine residues are identified
by vertical lines.
In the present study, we first expressed ST8Sia IV in insect cells
using a Baculovirus system. We found that ST8Sia IV produced in insect
cells or mammalian cells exists as a monomer and adds polysialic acid
to NCAM, although the enzyme preparation from the insect cells cannot
polysialylate itself ("autopolysialylation" (33, 34)). We then
determined by mass spectrometric analysis that disulfide bonds are
intramolecularly formed between the COOH terminus and sialylmotif L and
between sialylmotifs L and S. Moreover, site-directed mutagenesis
experiments demonstrated the absolute requirement of these disulfide
bridges and the position of the cysteine at the COOH terminus. These
results indicate that sialylmotifs L and S and the COOH-terminal region
are close to each other, and such a unique sterical structure is
important for the polysialylation activity of ST8Sia IV.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- Isolation of pcDNAI harboring ST8Sia IV cDNA, pcDNAI-ST8Sia IV, and a soluble form of ST8Sia IV fused to a signal peptide and the IgG binding domain of protein A, pcDNAI-A·ST8Sia IV, was described previously (18, 21). pcDNAI-ST8Sia II and pcDNAI-A·ST8Sia II were cloned as described previously (35). pIG-NCAM·IgG(VASE,MSD) encoding a soluble NCAM-140 fused with a hinge and constant regions of IgG was kindly provided by Dr. David Simmons at Oxford University (36). The vector, originally designated as pIG-NCAM, was found to contain both VASE and MSD sequences. Because such an NCAM(VASE,MSD) is unusual, a 2198-bp DNA sequence containing both VASE and MSD sequences was excised with HindIII and KpnI from pIG-NCAM(VASE,MSD) and was replaced with the corresponding HindIII/KpnI fragment (2050 bp) of NCAM-140 (17), resulting in pIG-NCAM. The obtained cDNA sequence was confirmed by nucleotide sequencing.
To construct cDNA encoding ST8Sia IV fused with FLAGF epitope or GFP protein, cDNA fragment encoding ST8Sia IV was amplified by PCR using T7 primer and 3'-primer (5'-CTGGTACCTGCTTTACACACTTTCC-3'; the KpnI site is underlined, and the rest of the sequence encodes nucleotides 1076-1060 of ST8Sia IV). The PCR product was digested with HindIII and KpnI and subcloned into the same sites of pFLAG-CMV-5b (Sigma) or pcDNA3.1-EGFP, resulting in pFLAG-ST8Sia IV or pcDNA3.1-ST8Sia IV·EGFP. Similarly, ST8Sia II cDNA fragment was amplified by PCR using T7 primer and 3'-primer (5'-TGGTACCGTGGCCCCATCGCACTGGCC-3'; the KpnI site is underlined, and the rest of the sequence encodes 1124-1105), and pFLAG-ST8Sia II and pcDNA3.1-ST8Sia II·EGFP were obtained. pcDNA3.1-EGFP was constructed from pEGFP-N1 (CLONTECH) by excising cDNA fragment with EcoRI/NotI digestion and cloned into the same sites of pcDNA3.1 (Invitrogen).
The cDNAs for the full-length enzymes fused with the FLAG epitope were expressed in HeLa cells, and all the lysates were dissolved in the sample buffer with or without 2-mercaptoethanol. FLAG-tagged enzymes were detected by Western blot analysis using anti-FLAG antibody (Sigma Chemical Co.) and the ECL Plus kit (Amersham Pharmacia Biotech).
Construction of Soluble ST8Sia IV and ST8Sia II Tagged with 6×His Peptide-- A vector for soluble form-containing 6×His peptide was first constructed as follows. A KpnI-HindIII fragment encoding a signal peptide from pcDNAI·A vector (21) and a HindIII-BamHI fragment of pcDNA3.1-HisB (Invitrogen) encoding for 6×His peptide were subcloned into pcDNA3.1-Hygro after digestion with KpnI and BamHI, resulting in pcDNA3.1-HSH (Hygro-Signal-6×His). A BamHI-XbaI fragment of pcDNAI-A·ST8Sia IV (nucleotides 118-1080) was subcloned into the same sites of pcDNA3.1-HSH, resulting in pcDNA3.1-His·ST8Sia IV, which harbors the coding region of a signal peptide, His tag, and catalytic domain of ST8Sia IV. Similarly, a PCR-amplified BglII-XhoI fragment of ST8Sia II (nucleotides 95-1125) (24) was placed into pcDNA3.1-HSH digested with BamHI and XhoI. His·ST8Sia IV and His·ST8Sia II released from transfected mammalian and insect cells were detected by Western blot analysis using anti-Xpress antibody (Invitrogen) and an ECL Plus kit.
Soluble ST8Sia IV Expression Using a Baculovirus System-- A BamHI-XbaI fragment of pcDNAI-A·ST8Sia IV (21) was subcloned into the same sites of pcDNA3.1-HisB. Using this construct as a template, cDNA encoding 6×His peptide and the same portion of ST8Sia IV as A·ST8Sia IV was amplified by PCR and subcloned into BamHI and XbaI sites of the pAcGP67A vector (PharMingen), which contains a gp67 secretion signal peptide driven by the Baculovirus polyhedrin promoter, resulting in pAcGP67A-His·ST8Sia IV. This transfer vector was used to obtain recombinant Baculovirus. High-Five insect cells, Trichoplusia ni (Invitrogen), were infected with the recombinant Baculovirus, and culture medium was collected 3 days after infection. His-tagged ST8Sia IV fusion protein, His·ST8Sia IV (BV), secreted from the insect cells was purified using ProBond Ni-beads (Invitrogen). His·ST8Sia IV (BV) eluted from Ni-beads was used as an enzyme source. For protein chemical analysis, the enzyme was concentrated by Microcon 30 (Millipore) and washed with 20 mM Tris-HCl, pH 7.4, three times. The recovered protein at a concentration of 0.5 µg/µl was used for disulfide mapping analysis as follows.
Liquid Chromatography/Electrospray Ionization-Tandem Mass Spectrometry (LC/ESI-MS/MS) Analyses-- A sample of the concentrated ST8Sia IV (~15 µg in 30 µl) was treated with a 20 times molar excess of PEO-maleimide-activated biotin and immediately denatured with 8 M urea. The sample was incubated for 60 min in the dark at room temperature (total volume, 45 µl). The concentration of urea in the sample was reduced to 2 M by addition of water. Trypsin or chymotrypsin (1/10, w/w ratio of the protease/protein) was then added, and the mixture (180 µl) was incubated overnight at 37 °C. A portion of the proteolytic digest was treated with N-glycanase overnight at 37 °C. N-glycanase was dissolved at a concentration of 200 units/ml in 100 mM sodium phosphate containing 25 mM EDTA, pH 7.2, and added to the proteolytic digest to a final concentration of 20 units/ml. The digests were fractionated using a capillary C18 column (150 × 0.18 mm; Nucleosil, 5-µm particle size) and analyzed on a Finnigan LCQ ion trap mass spectrometer with a modified electrospray ionization (ESI) source (37). The LC/MS analysis was conducted using a Hewlett-Packard 1050 high pressure liquid chromatography system coupled to the LCQ. The mobile phase was split before the injector by a Tee-connector, and a flow rate of 2 µl/min was established through the capillary C18 column. The sample was eluted by three-step linear gradient from 95% of the solution A (0.5% formic acid in water) and 5% of the solution B (0.5% formic acid in acetonitrile) in the first 10 min, 10-35% of the solution B in the next 40 min, and 35-40% of the solution B in the last 5 min. The LC/ESI-MS/MS analysis was performed using an automated data acquisition procedure, in which a cyclic series of three different scan modes was performed (35, 36). Data acquisition was conducted using the full scan mode (m/z 300-2000) to obtain the most intense peak (signal > 1.5 × 105 counts) as the precursor ion, followed by a high resolution zoom scan mode to determine the charge state of the precursor ion and an MS/MS scan mode (with a relative collision energy of 38%) to determine the structural fragment ions of the precursor ion. The resulting MS/MS spectra were then searched against a protein data base (Owl) by Sequest to confirm the sequence of tryptic or chymotryptic peptides. The details of the LC/ESI-MS/MS procedure were described elsewhere (38).
Deletion and Mutation of ST8Sia IV cDNA-- Five Cys residues in ST8Sia IV were mutated to Ala by the PCR. C142A was introduced by ligating two PCR-amplified fragments. 5'- and 3'-primers for the first PCR are T7 primer and 5'-CGGCCGGCGGTCTTAAACCTGCGATTCTTC-3'. For the second PCR, 5'-CTTTGCGCAGTTGTTGGAAATTCTGGCAT-3' (5'-primer) and a downstream primer containing the stop codon and XbaI site were used for the second PCR. After digestion with NaeI and FspI, these two PCR products are ligated at GCCGCA sequence containing Ala142 instead of Cys142 (the codon for Ala142 is singly underlined). Other mutations were made as follows: C156A (GAGGCA, generated by digestion with Ecl136II (GAGCTC) and FspI (TGCGCA)), C169A (AGGGCA, generated by digestion with StuI (AGGCCT) and FspI (TGCGCA)), C292A (TTTGCA, generated by digestion with DraI (TTTAAA) and FspI (TGCGCA)); single underlines denote Ala codons. C356A mutant was made as described below.
From the ST8Sia IV COOH terminus, two to four amino acids were removed by PCR using the 5'-primer 5'-GGGTCTCCCTCAGTTGTACAAAGAGCATTT-3' (the underlined sequence corresponds to nucleotides 577-597) and the 3'-primer containing a deletion followed by a stop codon and an XbaI site. The amplified fragment digested with PmlI and XbaI replaced wild-type sequences of pcDNAI-ST8Sia IV. Similarly, mutant forms of ST8Sia IV, in which Cys356 has been altered to Ala356, Cys354, Cys355, Cys357 or Cys358, or in which Thr353, Gly354, or Lys355 is mutated, were generated by the same PCR method using mutated 3'-primers.
Expression of Polysialic Acid on Cells Transfected with Mutated cDNAs-- cDNAs encoding membrane or soluble forms of wild-type and mutant enzymes were transfected into COS-1 cells or HeLa cells stably expressing NCAM using LipofectAMINE (Life Technologies) as described previously (23). Forty-eight h after transfection, the cells were fixed and stained with 12F8 (PharMingen), a rat monoclonal antibody specific to polysialic acid (39), followed by fluorescein isothiocyanate- or Rhodamine-conjugated goat immunoglobulins specific to rat IgM (Cappel). The same set of antibodies was used to analyze transfected cells by FACSort (Becton Dickinson), as described previously (23).
In Vitro Polysialyltransferase Assays--
COS-1 cells
were transfected with cDNAs encoding wild-type or mutant forms of
soluble enzymes. Forty-eight h after transfection, the culture medium
was changed to serum-free medium (Life Technologies), and the cells
were incubated for another 24 h. Soluble enzymes were collected
from the spent media using human IgG-Sepharose (Amersham Pharmacia
Biotech). The quantity of each enzyme bound to the beads was measured
by Western blot analysis using peroxidase-conjugated rabbit
immunoglobulins (Cappel) and an ECL Plus kit as described previously
(23). An equivalent amount of each mutant enzyme adsorbed to beads was
used for in vitro sialyltransferase assays in 50 mM sodium cacodylate buffer, pH 6.0, containing 2.5 mM MgCl2, 2.5 mM MnCl2,
1 mM CaCl2, 0.5% Triton CF-54, 2.4 nmol (0.7 µCi) of CMP-[14C]NeuNAc, and 10 pmol of NCAM·IgG.
After incubation, NCAM·IgG in the supernatant was obtained by brief
centrifugation and subjected to SDS-polyacrylamide gel electrophoresis
followed by fluorography as described previously (21, 23). For the
assay of autopolysialylation, the incubation mixture omitted
NCAM·IgG, and the radiolabeled enzymes were released from beads and
subjected to SDS-polyacrylamide gel electrophoresis followed by
fluorography (24). Similarly, His·ST8Sia IV (BV) secreted from insect
cells was used as an enzyme source under the same assay conditions.
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RESULTS |
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ST8Sia IV Synthesized in Insect Cells Polysialylates NCAM but
neither Autopolysialylates nor Forms a Homodimer--
To determine the
structure and function of ST8SiaIV, ST8Sia IV was produced in insect
cells as a soluble protein fused with 6×His peptide. Fig.
2A shows that ST8Sia IV
produced in the insect cells, His·ST8Sia IV (BV), migrated at the
same position regardless of the presence or absence of reducing agent.
Its molecular mass (~46 kDa) corresponds to that expected from the
amino acid sequence (41 kDa) plus glycosylation, indicating no dimer
was formed. To confirm that this is true also for an intact form of
ST8Sia IV and ST8Sia II, the entire sequence of ST8SiaIV or ST8Sia II
fused with FLAG tag peptide at the COOH terminus was expressed in HeLa cells. It was shown that ST8Sia IV or ST8Sia II fused with the FLAG tag
can form polysialic acid in transfected HeLa cells (data not shown).
Although a small amount of dimer was also formed in the absence of a
reducing agent, ST8Sia IV and ST8Sia II mainly existed as a monomer
(Fig. 2B). The negligible amount of dimers was probably due
to nonspecific aggregation, because this amount was increased when the
samples were frozen and heated in the sample buffer under milder
conditions (data not shown).
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As shown in Fig. 3A, ST8Sia IV
produced in the insect cells added as much polysialic acid to NCAM as
did ST8Sia IV produced in HeLa cells. ST8Sia IV produced in the insect
cells, however, failed to add polysialic acid to itself
(autopolysialylation) (Fig. 3B), most likely because ST8Sia
IV produced in insect cells contained only immature
N-glycans. Indeed, endoneuraminidase treatment did not
change the molecular mass of ST8Sia IV produced in the insect cells
(data not shown). Moreover, the size of this ST8Sia IV produced in
insect cells was smaller than that of the same enzyme produced in HeLa
cells (Fig. 2A), indicating the difference in
N-glycosylation between two cell systems. These results
combined demonstrate that ST8Sia IV can extensively form polysialic
acid on NCAM when autopolysialylation is not detectable (see also
"Discussion").
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N-Glycosylation of ST8Sia IV Synthesized in Insect Cells and
Mammalian Cells Utilizes the Same Glycosylation Sites--
Previously
it has been shown that Asn74 is the
N-glycosylation site where the majority of
autopolysialylation occurs (34). The absence of autopolysialylation
could thus occur if Asn74 is not utilized in the insect
cells. To determine whether or not N-glycan was attached to
Asn74, the ST8SiaIV sample used in the above experiments
was subjected to mass spectrometric analysis after tryptic digestion.
By liquid chromatography/electron spray ionization-tandem mass
spectrometry (LC/ESI-MS/MS) analysis, more than 86% of the amino acid
sequence predicted from the ST8Sia IV cDNA was confirmed using the
protein data base searching program Sequest. Four of the five peptides that contain N-glycosylation consensus sequences were not
detected, but a peptide containing Asn219 was detected.
After treatment with N-glycanase, however, the other
peptides containing a consensus N-glycosylation sequence (peptides containing Asn50, Asn74,
Asn119, Asn204, and Asn219) were
detected as peptides containing an Asp residue in place of the expected
Asn residue. This conversion is expected after N-glycanase
treatment. For example, the peptide (amino acid residues 59-82) that
contains the Asn74 (Asn Asp) residue was detected at
m/z = 895.8 (monoisotopic peak) for a triply
charged ion, which corresponds to the measured (M+H)+ = 2687.4 Da instead of the expected (M+H)+ = 2686.4 Da for a
peptide with Asn (Fig. 4A and
its inset mass spectrum). The MS/MS analysis of the triply
charged ion at m/z = 895.8 showed that
dominant product ions were generated from N-terminal
fragments, yn (n = 8-10, and 17),
and C-terminal fragments bn (n = 11, 13, and 21) (Fig. 4B). The assignment of MS/MS fragments is
based on Biemann's nomenclature (40). The mass difference (114.9 Da)
between y8 (m/z = 916.5) and y9 (m/z = 1031.4) provides unambiguous confirmation that the sequence contains
Asp74 in place of Asn74. These results combined
demonstrate that an N-glycan is attached to
Asn74 but not polysialylated. Similarly, the results
demonstrate that Asn50, Asn119, and
Asn204 are N-glycosylated, whereas
Asn219 is partially N-glycosylated (see Fig.
1).
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Identification of Free and Disulfide-bonded Cys Residues-- As seen in Fig. 1, ST8Sia IV and other members of the ST8Sia gene family are unique in having a cysteine residue at the COOH terminus, three cysteine residues in sialylmotif L, and one cysteine in sialylmotif S. In contrast, sialyltransferases of other families contain only one conserved cysteine residue in each sialylmotif L and S. These results suggest that the members of the ST8Sia gene family, including ST8Sia IV, may share unique disulfide bond structures. To determine the disulfide bond structures of ST8Sia IV, the enzyme was subjected to mass spectrometric analysis. ST8Sia IV purified from the insect cells was reacted with polyethylene oxide-maleimide biotin (PEO-maleimide biotin) to label any free Cys residues, denatured, and digested with trypsin or chymotrypsin under non-reducing conditions. The resultant peptides were separated by liquid chromatography and analyzed for peptides containing modified Cys residues by Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS) analysis. A doubly charged ion for the Cys169-containing peptide (amino acid residues 166-177) at m/z = 943.0 was detected. The MS/MS analysis of the ion specie at m/z = 943.0 conclusively demonstrated that Cys169 was conjugated to the PEO-maleimide biotin with a mass equivalent to that expected for a peptide with a biotinylated Cys residue (data not shown). None of the other Cys-containing peptides of ST8Sia IV was found to contain a biotinylated Cys residue.
To determine if Cys residues other than Cys169 participate
in disulfide bonds and determine how these residues are linked,
chymotryptic digests of non-reduced ST8Sia IV were subjected to
LC/ESI-MS/MS analysis. Calculations of all possible disulfide-linked
patterns were made, and the data generated from the LC/ESI-MS/MS
analysis of a non-reduced chymotryptic digest of ST8Sia IV were
searched for possible matches. The search identified a doubly charged
ion at m/z = 1082.9 and a triply charged ion
at m/z = 722.6, which correspond to the
predicted m/z values for a disulfide-linked peptide (Cys142-Cys292, Fig.
5A) containing amino acid
residues 140-152 and 292-298. The MS/MS analysis of the doubly
charged ion at m/z = 1082.9 produced dominant ions, which were generated from N-terminal fragments, yn (n = 3-5, and 7), from the
Cys292-containing peptide (amino acid residues 292-298)
and COOH-terminal fragments, bn (n = 5, 6, and 7), from the Cys142-containing peptide (amino
acid residues 140-152; Fig. 5B). Each mass of generated
peptides was consistent with the calculated mass, confirming the
sequence and disulfide structure of
Cys142-Cys292.
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The search for possible matches also identified a second
disulfide-linked peptide (Cys156-Cys356) that
was detected as both a doubly charged ion at
m/z = 1292.5 and a triply charged ion at
m/z = 862.4 (Fig.
6A). The MS/MS spectrum of the
doubly charged ion at m/z = 1292.5 showed
that the peptide contains disulfide-linked dipeptide containing amino
acid residues 153-165 and residues 350-359 (Fig. 6B).
These combined results demonstrate that ST8Sia IV contains two
disulfide linkages of Cys142-Cys292 and
Cys156-Cys356. In parallel, the above
non-reduced and biotinylated ST8Sia IV was reduced by dithiothreitol
and alkylated with iodoacetamide. LC/ESI-MS/MS analysis detected four
carbaminomethylated cysteine-containing (Cys142,
Cys156, Cys292, and Cys356)
peptides, supporting the above conclusion that these four cysteine residues are involved in disulfide bonds.
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Site-directed Mutagenesis of Cysteine Residues--
To determine
the roles of disulfide bond and Cys169, five cysteine
residues in the catalytic domain of ST8Sia IV were individually mutated, and mutant enzymes were expressed. The results demonstrated that mutation of either Cys142, Cys156,
Cys292, or Cys356 to Ala completely inactivated
the enzyme when assayed for both NCAM polysialylation and
autopolysialylation (Fig. 7). To exclude the possibility that this inactivation was caused by undesired disulfide pairing, cysteine residues involved in each disulfide bond
were mutated together (C142A/C292A and C156A/C356A). The results
confirmed the above conclusion that all of the disulfide bond
structures are crucial for the enzymatic activity (Fig. 7). A mutation
at Cys169 also reduced the enzyme activity; however, a
small detectable activity for NCAM polysialylation was found for this
mutation, whereas autopolysialylation activity was scarcely detected
for the same mutant enzyme (Fig. 7). These results combined together indicate that Cys142, Cys156,
Cys292, and Cys356 are important for the
catalytic activity of ST8Sia IV by forming disulfide bonds whereas
Cys169 is involved in its catalytic activity as a free
cysteine.
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Roles of COOH-terminal Sequence--
The above results
demonstrated that Cys356 close to the COOH-terminal end is
critical for the enzymatic activity. To determine if the other amino
acid residues in the COOH-terminal sequence are important, the
COOH-terminal sequence was either deleted or mutated. The results
demonstrated that removal of three amino acid residues from the CVKQ
terminus (IV-3 in Fig. 8) had almost no
effect on activity (Fig. 9B),
but deletion of Cys356 (ST8Sia IV-4 in Fig. 8) inactivated
the enzyme (Fig. 9C) consistent with the above result.
Mutating lysine or glycine or threonine residues adjacent to
Cys356 resulted in only modest decrease in the
polysialylation (compare IV-5+AC, IV-6+AAC, or IV-7+AAAC with IV-3 in
Figs. 8 and 9F).
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Interestingly, changing the position of Cys356 to 1
(IV-5+C),
2 (IV-6+C), +1 (IV-4+AC), or +2 (IV-4+AAC, see Fig. 8)
resulted in almost complete loss of polysialylation activity (Fig. 9,
D and E). These results indicate that there is a
strict requirement for the position of the Cys356 residue.
In all of these mutation experiments (shown in Figs. 7-9), none of the
mutated enzymes showed a detectable misfolding, because all of the
soluble mutated enzymes were secreted into the medium in almost
equivalent amounts.
The above results suggest the sterical structure formed by the
disulfide bond between Cys356 and Cys156 is
critical, but amino acid residues 357-359 may not be critical for the
enzymatic activity. To determine if the addition of a bulky protein to
the COOH end renders ST8Sia IV inactive, ST8Sia IV was fused with GFP
protein (Mr ~ 27 kDa) at the COOH terminus. HeLa cells transfected with control GFP was negative for polysialic acid (Fig. 9G). Surprisingly, the addition of GFP to ST8Sia
IV did not interfere with the enzymatic activity of ST8Sia IV (Fig. 9H). Identical results were obtained on ST8Sia II (Fig.
9I). In contrast to GFP in control cells (Fig.
9G), the ST8Sia IV·GFP or ST8Sia II·GFP was stained in
perinuclear regions, probably corresponding to the Golgi apparatus.
These combined results suggest that the amino acid sequence flanking
Cys356 is not directly involved in catalytic activity but
the sterical structure formed by the disulfide bonded
Cys356-Cys156 is critical for its activity.
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DISCUSSION |
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In the present study, we demonstrated that ST8Sia IV produced in T. ni insect cells can add polysialic acid to NCAM, although it cannot add polysialic acid to itself (autopolysialylation) (Fig. 3). Similarly, the C169A mutant enzyme catalyzed NCAM polysialylation but was barely capable of autopolysialylation (Fig. 7). Previously, it was shown that N-glycans of IgG secreted from T. ni cells lack sialic acid and contain only small amounts of galactosylated N-glycans (41). The majority of these N-glycans contain N-acetylglucosamine- or mannose-terminated N-glycans. We also showed that ST8Sia IV produced in T. ni cells contained an N-glycan attached to Asn74, where autopolysialylation preferentially takes place in mammalian cells (34). These combined results strongly suggest that the lack of autopolysialylation for ST8Sia IV produced in the insect cells is due to a lack of sialylated N-glycans that serve as an acceptor for the enzyme. Previously, Windfuhr et al. (42) reported that autopolysialylation is necessary for NCAM polysialylation, whereas Close et al. (34) reported that autopolysialylation is not necessary for NCAM polysialylation. Our present study unequivocally demonstrated that ST8Sia IV can add polysialic acid to NCAM in the absence of detectable autopolysialylation.
In the present study, we also determined the disulfide bond structures
of ST8Sia IV using improved LC/ESI-MS/MS analysis (37, 38). Our results
demonstrated that the cysteine residue at the COOH-terminal end forms a
disulfide bond with the second cysteine residue of sialylmotif L and
that the second disulfide bond is formed between the first cysteine at
sialylmotif L and the cysteine at sialylmotif S. These disulfide bonds
are intramolecularly formed, because almost no homodimer was detected
(Fig. 2). These disulfide bonds thus form a unique structure of ST8Sia
IV as depicted in Fig. 10 (middle
graphic). These disulfide bond structures are imperative for the
enzymatic activity, because replacement of any of these cysteine
residues with alanines resulted in inactivation of the enzyme. This
finding is consistent with a recent report on ST8Sia IV derived from
mutant Chinese hamster ovary cells, showing that a Cys356
to Phe356 mutation inactivated the enzyme (42). On the
other hand, the third cysteine, Cys169, in sialylmotif L is
present as a free cysteine as shown by mass spectrometric analysis.
This cysteine is also involved in the enzymatic activity, because the
mutation of this cysteine169 to alanine reduced the
enzymatic activity. This result is consistent with a previous report
that at least one free cysteine residue is critical for polysialylation
(43).
|
In the present study, we also demonstrated that the position of Cys356 at the COOH-terminal end is an absolute requirement and one residue movement of Cys356 to the NH2- or COOH-terminal site abolishes polysialylation activity. In contrast, the removal of three residues from Val357-Lys358-Gln359-COOH did not impair the enzymatic activity. Replacement of Thr353, Gly354, and Lys355 with Ala did not impair the enzymatic activity either (Figs. 8 and 9). These results indicate that disulfide bond formation by Cys356 is highly constrained in terms of its position, whereas flanking amino acid sequences may not be directly involved in the enzymatic activity. These conclusions can be supported by the fact that the amino acid sequences flanking the conserved cysteine at the COOH terminus are not conserved among five members of the ST8Sia gene family (16-20, 44-46). For example, human ST8Sia III contains only one additional amino acid, alanine after the cysteine residue (45). These results contrast with the previously obtained results on fucosyltransferase V and N-acetylglucosaminyltransferase V; removal of one and five residues from their COOH terminus abolished the enzymatic activity, although neither enzyme contains a cysteine residue at its COOH terminus (47, 48). Moreover, the results obtained by site-directed mutagenesis of ST8Sia IV was corroborated by the fact that the addition of GFP protein at the COOH end of ST8Sia IV does not interfere with the enzymatic activity. These results combined suggest that the amino acid sequence flanking Cys356 may not be directly involved in the catalytic activity of ST8Sia IV, but the sterical structure formed by the disulfide bonds is critical for its activity. The results also provide a useful tool in understanding the roles of ST8Sia IV, because we can determine if the phenotype of those cells expressing ST8Sia IV, detected by GFP produced, differ from those which do not express ST8Sia IV.
Disulfide bond structures found for ST8Sia IV are unique in a way
that the amino acid sequence downstream from sialylmotif S to the COOH
terminus is in close proximity with sialylmotif L (Fig. 10). This
sterical structure is most likely shared by all members of the ST8Sia
gene family, because these four cysteine residues are conserved in
these enzymes (see Fig. 1). Another member of the ST8Sia gene family,
ST8Sia I, has been shown to add continuously one or more 2,8-linked
sialic acids to glycolipids (44). The oligosialylated glycolipids are
also synthesized by ST8Sia V (46). ST8Sia III as well as ST8Sia II and
IV were shown to polysialylate itself (24). All
2,8-sialyltransferases thus share the ability to add more than one
2,8-linked sialic acid to specific acceptor molecules. Such common
catalytic activity is observed in only
2,8-sialyltransferases,
possibly because common disulfide bond structures are shared by this
family (see Fig. 10). Sialyltransferases of other gene families, ST3Gal
I for example (49), contain only one conserved cysteine in sialylmotif L, which corresponds to the first cysteine in sialylmotif L in ST8Sia
IV, and another conserved cysteine in sialylmotif S. If these two
cysteine residues are linked to each other as shown in ST8Sia IV, this
disulfide bridge would bring sialylmotifs L and S into a close
proximity (see Fig. 10). This hypothesis is consistent with the
findings that both sialylmotifs L and S are involved in binding to
CMP-NeuNAc (30, 31). However, these sialyltransferases lack two
additional cysteine residues conserved in the ST8Sia gene family,
suggesting that the COOH-terminal region of these sialyltransferases
may not be close to sialylmotif L. It is still possible that these
sialyltransferases may form a similar structure without a disulfide
bond. But such a structure must be different from that constrained by a
disulfide bond shown in ST8Sia IV.
In the present study, we found that ST8Sia II and ST8Sia IV form a
negligible amount of disulfide-bonded dimer, if any. Previous studies
suggest that homodimers and possibly heterodimers of
1,4-galactosyltransferase or
N-acetylglucosaminyltransferase I may correlate with
its Golgi localization (50, 51). Nillson et al. (52)
proposed that the medial Golgi enzymes,
-mannosidase II and
N-acetylglucosaminyltransferase I, form heterodimers through
kin recognition that confers Golgi localization. In all of those
studies, the formation of hetero- or homodimers through a disulfide
bond was not addressed. Chen et al. (53), on the other hand,
demonstrated that one isoform of ST6Gal I forms disulfide-linked
homodimers, which stay in the Golgi. The formation of disulfide-linked
homodimers has been also reported for
1,2-fucosyltransferase, GM2
synthase, and
1,3-glucuronyltransferase (51, 54, 55). For GM2
synthase, homodimers are formed in anti-parallel orientation, and such
a dimeric formation is apparently essential for its activity (54). In
contrast,
1,3-glucuronyltransferase that adds the first glucuronic
acid in proteoglycan synthesis (55) forms a functional homodimer in
parallel orientation through a cysteine in the stem region. Different
from these enzymes, it was shown by crystallographic studies that
1,4-galactosyltransferase and
N-acetylglucosaminyltransferase I do not form homodimers
through disulfide bridges and only intramolecular disulfide bridges are formed in these enzymes (56, 57). Our results indicate that ST8Sia IV
belongs to this third group, which does not form homodimers through
disulfide bridges. It will be significant to determine if other
sialyltransferases belong to any of the above three groups in terms of
disulfide bond structures and if such a disulfide bond structure is
related to the mode of its catalytic activity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. David Simmons for pIG-NCAM·IgG(VASE,MSD), Dr. Rita Gerardy-Schahn for pCDM8-NCAM-140, Dr. Edgar Ong for critical reading of the manuscript, and Joseph P. Henig and Risa Tabata for organizing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Research Grants R01 CA33895 (to M. F.) and P20 RR11805 (RIMI) and by National Science Foundation Grant MCB-9816780 (to B. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: The Burnham
Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3144; Fax: 858-646-3193; E-mail: minoru@burnham.org.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M100576200
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ABBREVIATIONS |
---|
The abbreviations used are: NCAM, neural cell adhesion molecule; PCR, polymerase chain reaction; GFP, green fluorescence protein; EGFP, enhanced green fluorescence protein; LC/ESI-MS/MS, liquid chromatography/electrospray ionization-tandem mass spectrometry; PEO, polyethylene oxide; bp, base pair(s).
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