From the Department of Anatomy and Cell Biology, College of
Medicine, University of Florida, Gainesville, Florida 32610-0235 and the Department of Biochemistry, Imperial College,
London SW7 2AY, United Kingdom
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ABSTRACT |
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SKP1 is involved in the ubiquitination of certain
cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an
oligosaccharide containing Fuc and Gal, which is unusual for a
cytoplasmic or nuclear protein. To establish how it is glycosylated,
SKP1 labeled with [3H]Fuc was purified to homogeneity and
digested with endo-Lys-C. A single radioactive peptide was found after
two-dimensional high performance liquid chromatography. Analysis in a
quadrupole time-of-flight mass spectrometer revealed a predominant ion
with a novel mass. Tandem mass spectrometry analysis yielded a set of
daughter ions which identified the peptide and showed that it was
modified at Pro-143. A second series of daughter ions showed that
Pro-143 was hydroxylated and derivatized with a potentially linear
pentasaccharide, HexHex
Fuc
Hex
HexNAc
(HyPro). The
attachment site was confirmed by Edman degradation. Gas
chromatography-mass spectrometry analysis of trimethylsilyl-derivatives
of overexpressed SKP1 after methanolysis showed the HexNAc to be
GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1
and from a fucosylation mutant, followed by matrix-assisted laser
desorption time-of-flight mass spectrometry analysis, showed that the
sugar chain consisted of D-Galp
1
6-D-Galp
1
L-Fucp
1
2-D-Galp
1
3GlcNAc.
Matrix-assisted laser-desorption time-of-flight mass spectrometry
analysis of all SKP1 peptides resolved by reversed phase-high
performance liquid chromatography showed that SKP1 was only partially
hydroxylated at Pro-143 and that all hydroxylated SKP1 was completely
glycosylated. Thus SKP1 is variably modified by an unusual linear
pentasaccharide, suggesting the localization of a novel glycosylation
pathway in the cytoplasm.
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INTRODUCTION |
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SKP1 is found in a multiprotein complex with cullin (a cdc53 homologue) and an F-box-containing protein to form the SCF complex, named as an acronym of the participating proteins. When this complex contains an E2 enzyme, it is responsible for ubiquitinating various target proteins, depending on the identity of the F-box protein. Targets for subsequent degradation identified in Saccharomyces cerevisiae include cell cycle proteins such as the S-phase kinase inhibitor SIC1 and G1 cyclins, and proteins specific to the nutrition of the cell (1-4). The SCF complex has also been implicated in phosphorylation of kinetochore proteins (5), and another distantly related complex affects mRNA metabolism (6). An SCF complex with Cyclin A and Cdk2 has been detected in mammalian cells, and its abundance appears increased in transformed cells (7, 8). SKP1 itself is abundantly and dynamically expressed in the mouse embryo (9) and central nervous system including postmitotic neurons (10) and at very high concentrations in the inner ear organ of Corti (11, 12), where it comprises up to 5% of total protein in the cytoplasm. The expression of several SKP1 genes in plants appears to be governed by morphogenetic boundaries (13). Thus SKP1 is expressed ubiquitously in eukaryotes, and its role may be to facilitate the selection of other proteins for specific posttranslational modification.
Binding of SKP1 to different proteins may be regulated structurally because it is encoded by multiple genes in multicellular organisms (1, 11, 14-16). SKP1 structure is also altered by glycosylation in Dictyostelium discoideum (17, 18), which may regulate its activity. Because complex glycosylation of cytoplasmic/nuclear proteins is unusual, we embarked on a study to establish the structure of the carbohydrate modification as a first step in investigating its function.
To determine the structure and site of attachment of the previously described SKP1 fuco-oligosaccharide(s) (17, 18), mass spectrometric approaches were employed. Key to the success of the this methodology was the newly developed Q-TOF MS1 (19, 20), which permitted MS-MS studies to be performed on the picomole quantities of material available from native sources. This recently designed instrument comprises a quadrupole with collision cell followed by an orthogonal acceleration TOF analyzer that confers a high degree of sensitivity and accuracy to the mass measurements. The ability of this instrument to sequence both the peptide and oligosaccharide chains of an SKP1 fucoglycopeptide suggested that it consists of a linear pentasccharide attached to hydroxylated Pro-143. The sugar sequence was established from exoglycosidase studies using MALDI-TOF MS and sugar analyses on genetically overexpressed material. The protein attachment site was then confirmed by Edman degradation.
These results reinforce the model that complex O-linked glycosylation occurs in the eukaryotic cytoplasmic compartment. Whereas there has been much circumstantial evidence to support this model (21), it has remained controversial (22). In contrast, simple glycosylation, in the form of GlcNAc O-linked to residues of Ser or Thr, is well established in this compartment (23). The sugar structures that have been described to date on cytoplasmic/nuclear proteins are generally distinctive from those produced in the secretory pathway, suggesting that there may be fundamental differences in the biogenesis and function of these modifications.
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EXPERIMENTAL PROCEDURES |
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Construction of Strain HW120--
A full-length cDNA from
the fpa1 gene with a decapeptide encoding the human
c-myc epitope (24) inserted between codons 161 and 162 near
the C terminus was prepared by PCR and cloned into pT7Blue (Novagen).
The cDNA was subcloned into pVEIIATG (25) under the control of
the inducible discoidin promoter and the actin 8 terminator. The
plasmid was transformed into D. discoideum by a
CaPO4 precipitation method and selected in the presence of 15 µg/ml G418 (26). Strain HW120 was a clone that produced
SKP1-c-myc at high levels after growth in 120 µg/ml G418,
based on Western blot analysis with monoclonal antibody 9E10 against
the c-myc epitope. SKP1-c-myc contained two
missense mutations, T194C and A305G resulting in the amino acid
substitutions I34T and D71G, introduced by the PCR reaction and
confirmed by MS analysis of its peptides (data not
shown).
Purification of SKP1-- D. discoideum strains Ax3, HL250, or HW120 were grown to stationary phase in HL-5 medium (17). Cells were filter-lysed, and an S100 fraction was prepared by ultracentrifugation (17). SKP1 from Ax3 and HL250 were fractionated into pools-I and -II by DEAE-anion exchange chromatography (14); SKP1-c-myc from strain HW120 eluted after pool-II. Each pool was subsequently purified by phenyl-Sepharose and monoclonal antibody 3F9 affinity chromatographies (14). For metabolic labeling, a 200-ml culture of Ax3 cells was grown for three generations in 0.05 mCi/ml [3H]Fuc in HL-5. SKP1 was isolated in the same manner except the phenyl-Sepharose step was omitted, and purified radiolabeled SKP1 was pooled with unlabeled SKP1. In some cases, SKP1 was reduced and carboxamidomethylated (14), and further purified on a C2/C18 (PC 3.2/3) RP-HPLC column on an Amersham Pharmacia Biotech SmartSystem HPLC (14). The peak that contained >80% of total SKP1 was examined further. Recombinant SKP1 (fpa1) containing an N-terminal oligo-His tag was isolated from Escherichia coli as described (27) and further purified on an RP column.
Monosaccharide Composition Analysis-- 2 nmol of mannitol was added as an internal standard to 1.5 nmol of SKP1-c-myc. The sample was subjected to methanolysis, re-N-acetylation, and TMS-derivitization as described (28) and analyzed in splitless mode on a 0.32 mm x 30 m SPB-1 column (Supelco) on a Shimadzu QP-5000 GC/MS workstation. Peaks were analyzed with selected ion monitoring at m/z 204 for the neutral sugars and m/z 173 for the N-acetylated amino sugars.
Purification of Glycopeptides-- Carboxamidomethylated SKP1 was digested with endo-Lys-C from Achromobacter lyticus (Wako Chemicals, Richmond, VA) in the presence of 2 M urea in 0.2 M Tris-HCl, pH 7.4, using an enzyme:substrate ratio of 1:200 (mol:mol), at 30 °C for 18 h. Peptides from metabolically labeled SKP1 were fractionated on a Superdex Peptide HR10/30 column (Amersham Pharmacia Biotech) in 6 M urea in 50 mM NaCl, 50 mM Tris-HCl, pH 7.2, on an LKB GTi HPLC system at 0.5 ml/min. Radioactive fractions were applied to a 4.6 × 150 mm, 3.5-µm particle C8 column (Zorbax) and eluted with a gradient of 5% (v/v) MeCN in 0.1% (v/v) trifluoroacetic acid to 40% MeCN in 0.085% trifluoroacetic acid at 1 ml/min. Peptides (20 pmol) were subjected to Edman degradation on an ABI model 494 Procise sequenator. Nonradiolabeled peptides were fractionated directly on the C8 or a C2/C18 (PC 2.1/10; Amersham Pharmacia Biotech) RP-column.
MALDI-TOF MS--
Samples were mixed with an equal volume of
saturated -cyano-4-hydroxycinnamic acid (Aldrich) in 70% MeCN. 1 µl (2-3 pmol) was deposited on a sample plate and air dried. Spectra
were collected on a PerSeptive Biosystem Voyager RP MALDI-TOF MS
operated in the positive ion, linear mode.
Q-TOF Mass Spectrometry-- Samples from RP fractions were introduced directly into the Q-TOF MS (Micromass, UK) via a nanospray device (19, 20). Primary and secondary ion spectra were collected in the positive ion mode.
Exoglycosidase Digestion of SKP1 Glycopeptides-5--
15 pmol of
glycopeptide from RP fractions were partially dried by vacuum
centrifugation, diluted to a final volume of 2.5 µl with the
glycosidase preparation, incubated for 18 h at 37 °C, and
processed for MALDI-TOF MS. Nonsusceptible substrates yielded no new
ions after digestion. Green coffee bean -galactosidase (Gal
1
4>2,3
6) from Boehringer Mannheim was further purified as described (29) and used at 10 milliunits/µl in 50 mM
ammonium phosphate, pH 6.0. Recombinant
-galactosidase (Gal
1
3)
from Glyko (Novato, CA) was used at 10 microunits/µl in 20 mM ammonium phosphate, pH 6.0. Xanthomonus
manihotis
-galactosidase (Gal
1
3/6) from New England
Biolabs was used at 10 microunits/µl in 20 mM ammonium
phosphate, pH 6.0. Sweet almond
-glucosidase (Glu/Gal/Fuc
) from Boehringer Mannheim was used at 10 milliunits/µl in 20 mM ammonium phosphate, pH 5.0. Bovine kidney
-fucosidase
(Fuc
1
2/3/4/6) from Boehringer Mannheim was used at 2.5 milliunits/µl in 20 mM ammonium phosphate, pH 5.0. X. manihotis
-fucosidase (Fuc
1
2) from New England
Biolabs was used at 100 milliunits/µl together with purified green
coffee bean
-galactosidase in 50 mM ammonium phosphate,
pH 6.0. Bovine kidney
-galactosidase (Gal
1
3/4>6) from Oxford
GlycoSciences (Abingdon, UK) was used at 4 milliunits/µl in 25 mM ammonium acetate, pH 4.0. Recombinant
-galactosidase (Gal
1
3/6) from Glyko was used at 4.8 milliunits/µl in 20 mM ammonium phosphate, pH 5.0. X. manihotis
-galactosidase (Gal
1
3) from New England Biolabs was used at
4.8 milliunits/µl in 20 mM ammonium phosphate, pH 5.0. Jack bean
-HexNAcase (GlcNAc/GalNAc
) from V-Labs was used at
6.5 milliunits/µl together with bovine kidney
-galactosidase in 20 mM ammonium phosphate, pH 5.0.
Mild Acid Hydrolysis of the Fucoglycopeptide-- The peptide fraction was partially dried in a vacuum centrifuge, reconstituted in 10 µl of 0.05 M trifluoroacetic acid, and incubated at 95 °C for up to 6 h.
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RESULTS |
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Isolation of the SKP1 Fucoglycopeptide-- To investigate the glycosylation of SKP1, normal cells (strain Ax3) were metabolically labeled with [3H]Fuc, and SKP1 was purified to homogeneity. Reduced and alkylated SKP1 was digested with endo-Lys-C and fractionated on a Superdex peptide gel filtration column (Fig. 1A). Radioactivity eluted as a single peak. When fraction 23 was separated on a C8 RP-HPLC column, radioactivity again eluted in a single peak, which was centered at fraction 35, did not absorb at 280 nm, and contained 30% of the original radioactivity (Fig. 1B). These results suggested that SKP1 contained only a single fucoglycopeptide.
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Q-TOF MS Analysis-- A parallel experiment using nonradioactive Fuc yielded material for MS analysis. The HPLC fraction (number 35) containing the putative fucoglycopeptide was subjected to tandem mass spectrometry on a Q-TOF mass spectrometer. Analysis of a few picomoles in the MS-only mode gave a major [M+3H]3+ signal at m/z 829.42 (Fig. 2, including inset). Note the resolved natural abundance 13C isotopes at m/z 829.70 and 830.03 showing that this signal is triply charged. It therefore derives from a molecule of mass 2485 Da. A series of doubly charged ions was also apparent in the spectrum, separated by sugar mass differences (m/z 1244, 1163, 1081, 1008, and 927). The ion at m/z 1244 is the [M+2H]2+ ion corresponding to the [M+3H]3+ at m/z 829.42, and the mass differences from this correspond to intervals of Hex, Hex, Fuc, and Hex, respectively.
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MALDI-TOF MS Analysis-- MALDI-TOF MS of the fucoglycopeptide provided corroborative evidence for the carbohydrate sequence predicted from the Q-TOF experiments. The MALDI spectrum contained a major M+H+ signal at m/z 2487 (Fig. 5A) which was equivalent to the triply charged ion at m/z 829.70 in the Q-TOF spectrum. In addition, a series of low abundance ions were observed at m/z 2326, 2164, 2017, 1855, and 1652 consistent with sequential loss of Hex, Hex, Fuc, Hex, and HexNAc, respectively. The low abundance ions appeared to be fragmentation products of the m/z 2487 ion resulting from similar frequency of scission of each of the glycosidic linkages (31) rather than incompletely glycosylated species, because less glycosylated peptides eluted later from the RP column (see below). The m/z 2487 ion was not seen in a similar analysis of RP-fractionated peptides (see below) of recombinant SKP1 isolated from E. coli (data not shown), showing that it was specific to the protein expressed in Dictyostelium.
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Sugar Composition Analysis-- To determine its monosaccharide composition, SKP1 was isolated from the S100 fraction of a D. discoideum strain (HW120) genetically modified to overexpress, under the control of the inducible discoidin promoter, a form of the protein with a c-myc-epitope tag near its C terminus. The sugars of purified SKP1-c-myc were examined by GC/MS after methanolysis, re-N-acetylation, and formation of TMS derivatives. To achieve the sensitivity required to analyze 1.5 nmol of SKP1-c-myc, splitless injection and selected ion monitoring were employed. Fuc (0.33 nmol), Gal (0.70 nmol), and GlcNAc (0.32 nmol) were the three most abundant sugars detected, and their low levels indicated that overexpressed SKP1 was under-glycosylated. No GalNAc was detected. Fuc, Gal, and Xyl were previously detected after 2 M trifluoroacetic acid hydrolysis (18), but GlcNAc was not. The stronger acid conditions of methanolysis were probably required to liberate GlcNAc from its linkage with HyPro, and Xyl is not found in the fucoglycopeptide according to the MS results. Thus the GC/MS data showed that the deoxyHex residue identified by MS was L-Fuc, as suggested by metabolic labeling, and showed that all three Hex residues were Gal and the HexNAc was GlcNAc.
Exoglycosidase Digestions--
To assign linkages, the
fucoglycopeptide was treated with exoglycosidases and analyzed by
MALDI-TOF MS. The mass of the glycopeptide was reduced by two Gal
residues (to m/z 2163) after treatment with the
nonspecific green coffee bean -D-galactosidase (Fig. 5C) but was not altered by nonspecific bovine kidney
-galactosidase, sweet almond
-glucosidase, or bovine kidney
-fucosidase. Furthermore, one Gal residue was susceptible to removal
by X. manihotis
1
3/6-galactosidase (Fig.
5B) but not recombinant Glyko
1
3-galactosidase,
showing that it was
1
6-linked. Fuc was susceptible to removal
(yielding m/z 2017) only after removal of the
-linked Gal residues, as shown by double enzyme digestion
experiments employing the
-galactosidase and either nonspecific
bovine kidney
-fucosidase or X. manihotis
1
2-L-fucosidase (Fig. 5D). Because Fuc
does not contain a 6-linkage site, this means that Fuc was capped by a
Gal
1
6Gal disaccharide, and the disaccharide was
-linked to
L-Fuc. These results indicate that the outer sugars form a
linear trisaccharide, Gal
1
6Gal
1
Fuc
1
2, in accord with
the MS data. A time course MALDI-TOF MS analysis during mild acid
hydrolysis of the fucoglycopeptide yielded a mass decrease of two Hex
residues and one deoxyHex residue (m/z 2487 to
m/z 2017) with no intermediates detected, as also
seen in the corresponding Q-TOF experiment (see above), confirming that
the outer Gal
-linked residues were attached to the acid-labile internal Fuc residue, whose more rapid release resulted in the simultaneous loss of all three sugars.
Glycosylation Heterogeneity--
Detection of the unmodified
peptide in the fucosylation mutant suggested that hydroxylation of
Pro-143, the first step in the glycosylation pathway, is rate-limiting
and possibly regulatory. Similarly, MALDI-TOF MS analysis of RP
fractions from endo-Lys-C released peptides from pool-I of SKP1 from
normal cells yielded ions corresponding only to the
pentasaccharidepeptide (m/z 2487) and
unmodified peptide139-152 (m/z 1637) (data not
shown). It remains to be determined whether Pro-143 hydroxylation
occurs on products of both SKP1 genes, as each are present in pools-I and -II, as shown previously by Edman degradation (14) and confirmed here by MS (data not shown). To characterize the glycosylation pathway
further, SKP1-c-myc was expressed at an elevated level in
strain HW120. Endo-Lys-C-generated peptides were fractionated by
RP-HPLC and analyzed by MALDI-TOF MS, yielding abundant
[M+H]+ ions of 2487, 2327, 2165, 1652, and 1636 in
successive fractions (data not shown). These corresponded to multiple
glycoforms, including the full-length pentasaccharide, the
pentasaccharide minus one or two of the outer
-linked Gal residues,
the hydroxylated but unglycosylated peptide, and the unmodified
peptide. These findings were consistent with the monosaccharide
composition results shown above. 1) Fuc and GlcNAc were present at
equal levels, as expected because no mono- and disaccharide
intermediates were detected. 2) The Gal:Fuc ratio was 2.1, indicating
that there is on average one
-linked Gal (the other being
-linked), which correlated with the detection of glycopeptides
containing 0, 1, or 2 outer Gal residues. 3) Only 22% of the protein
was glycosylated, which correlated with the high levels of unmodified
and hydroxylated forms of peptide 139-152 detected. The results
indicated that, secondary to Pro hydroxylation, attachment of the
reducing terminal GlcNAc and the outer
-linked Gal residues were the
next most rate-limiting, suggesting that the enzymes that add these
sugars may potentially regulate the structure of the Pro-143 glycan at other stages of the life cycle.
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DISCUSSION |
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The mass spectrometric analyses suggested that the SKP1 glycan
consists of a linear pentasaccharide attached to a HyPro at position143. The attachment site was confirmed by Edman degradation. The exoglycosidase studies and sugar analyses confirmed the linear model and showed the sequence to be
D-Galp1
6-D-Galp
1
L-Fucp
1
2-D-Galp
1
3-D-GlcNAc
HyPro-143. Substantially greater amounts of the fucoglycopeptide will be necessary
to determine the linkage position of the second
-linked Gal residue,
and the configuration of the GlcNAc
HyPro linkage. The core
trisaccharide, Fuc
1
2Gal
1
3GlcNAc, is equivalent to blood
group H (type 1) expressed by mammalian cells (32). Although internal
Fuc linkages have been found in glycoproteins (33, 34), the outer
Gal
1
6Gal
1
Fuc cap structure has not been previously described. However, this sugar chain is not immunogenic in mice (18),
unlike other Dictyostelium sugar protein conjugates (35, 36), suggesting that a similar structure may be expressed in mammals.
The linkage amino acid, hydroxyproline, is possibly 4-hydroxylated based on the specificity of a SKP1:UDP-GlcNAc GlcNAc-transferase activity that has been detected and partially
purified.2 4-hydroxylation is
phylogenetically ubiquitous and 4-OH-Pro has been found to be
derivatized with Ara or Gal in plants and algae (37, 38), but
substitution by GlcNAc has not been described previously. The
double-negative charge previously attributed to the sugar moiety after
attempted
elimination (17) may have resulted from base-catalyzed
scission of the adjacent E-E peptide bond, as sugar
HyPro linkages
are known to be alkali-resistant (37, 38). GlcNAc was not previously
detected in SKP1 (18), probably because methanolysis is more effective
than 2 M trifluoroacetic acid in causing its release from
HyPro.
The aforementioned structures homologous to the SKP1 oligosaccharide are synthesized in either the rER or the Golgi apparatus and then expressed on the cell surface or secreted. However, SKP1 is not secreted, but rather is located and functions in the cytoplasm and nucleus (1-13, 17, 18). This implies that known enzymes directing the synthesis of the homologous structures would not be accessible to SKP1, unless SKP1 transiently visits the lumen of the secretory pathway.
Current evidence suggests that SKP1 is partially modified in the
cytoplasm by a novel biosynthetic pathway. The enzyme which adds Fuc is
likely to be the previously characterized cytosolic fucosyltransferase
(cFTase). The cFTase fucosylates SKP1 in vitro with a
submicromolar Km (27) and catalyzes formation of the
same Fuc linkage on the same acceptor disaccharide (27, 39), as the
present results show occur naturally in SKP1. The cFTase is
likely to reside in the cytoplasmic compartment of the cell as it was
purified from the cytosolic fraction, and its submicromolar Km for GDP-Fuc is more characteristic of
cytoplasmic compared with Golgi glycosyltransferases (27). Recent
studies on Pro hydroxylase and GlcNAcTase activities that modify
overexpressed SKP1-c-myc and synthetic peptides suggest that
they also reside in the cytoplasmic compartment.3 These
enzymes may constitute a hitherto unrecognized complex pathway of
O-glycosylation localized in the cytoplasm, not the secretory pathway, that attaches sugars to SKP1 incrementally rather
than en bloc. Although glycosylation heterogeneity was not
observed in SKP1 from cells at the end of the growth phase, accumulation of incompeletely glycosylated chains in the overexpression strain raises the possibility of the expression of glycoforms at
Pro-143 at other stages of the life cycle.
Pro-143 is located within the highly conserved C-terminal domain of the SKP1 amino acid sequence (14). The C-terminal domain sequences are identical in the two Dictyostelium SKP1 genes, and a Pro at the equivalent position of Pro-143 is present in each of the numerous yeast, fungal, plant, and plant viral genes cloned to date. Of the nine SKP1 genes suggested by DNA sequencing data to exist in Caenorhabditis elegans (14), three are highly homologous to Dictyostelium SKP1 in this region, and two of these have the equivalent of Pro-143. Thus this key Pro residue may be modified in other organisms as well. The known mouse, guinea pig, and human cDNAs encode nearly identical proteins (7, 11, 15, 16) and lack this Pro residue, despite a high degree of similarity (>80%) with the Dictyostelium sequence in the C-terminal domain. Other mammalian SKP1 loci (11, 15, 16) may contain the equivalent of Pro-143, or SKP1 may be modified on a second, nearby TPEE sequence motif like that containing Pro-143.
The structural evidence shown here adds SKP1 to the short list of
examples that complex glycosylation does in fact occur on cytoplasmic
and nuclear proteins, as has often been postulated based on indirect
evidence (21, 22). It has been well documented in the past decade that
many cytoplasmic/nuclear proteins are monoglycosylated by GlcNAc on Ser
or Thr residues. However, the only generally accepted examples of
oligoglycosylation are an incompletely defined, pan-eukaryotic
Glc-1-PO4 modification of phosphoglucomutase (40), also
known as parafusin, and glycogenin, the primer for glycogen (41). The
novel GlcNAcHyPro linkage in SKP1 is distinct from the
GlcNAc
Ser/Thr (23), Glc
Tyr (41), and possible Man
Ser/Thr (40)
linkages found in the other cytoplasmic/nuclear proteins, and has not
been detected on proteins modified in the secretory pathway. Though
other proteins in the cytoplasmic fraction appear to be metabolically
labeled by [3H]Fuc, SKP1 appears to be a major recipient
of cFTase-mediated fucosylation both in vivo and in
vitro (17, 18). Further studies are required to determine whether
the other fucoproteins in this fraction reside in the
cytoplasmic/nuclear compartment prior to cell lysis. The availability
of the newly designed Q-TOF MS is expected to make it more practical to
investigate the posttranslational modifications of other lower
abundance intracellular proteins, which must be isolated from natural
sources to analyze their posttranslational modifications.
Cytoplasmic glycosylation can have dramatic consequences, as
highlighted by a Clostridial enzyme toxin that applies
GlcNAc to a specific Thr residue of cytoplasmic rho and cdc42 proteins resulting in major effects on the actin cytoskeleton (42). The function
of the SKP1 pentasaccharide modification is not likely to involve
competition with phosphorylation as proposed for the simple GlcNAc
monosaccharide modification of many cytoplasmic/nuclear proteins (23).
Instead, it may serve as a ligand for a cytoplasmic/nuclear carbohydrate-binding protein (43, 44) or as a steric shield. The
observation that the pentasaccharide modification appears to be
completed on all pool I and pool II SKP1 proteins whose Pro-143
residues are hydroxylated supports the model that it is a ligand for a
receptor, which must be structurally rich and constant. SKP1
subpopulations differing with respect to the
pentasaccharideHyPro modification might vary in their ability to
interact with specific F-box containing proteins, thereby potentially
regulating ubiquitination or phosphorylation of selected target
proteins in response to e.g. changes in the nutritional,
differentiation, or cell cycle status of the cell (1-13). The new
knowledge of glycan structure and attachment now renders the function
of SKP1 glycosylation susceptible to genetic investigation.
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ACKNOWLEDGEMENTS |
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Mr. E. Segura and Dr. D. Rentz at the University of Florida ICBR Protein Chemistry and Glycobiology Core Laboratories are acknowledged for the Edman degradation and monosaccharide analyses. H. van der Wel provided assistance in purifying the fucoglycopeptide, and Dr. N. Denslow of the Protein Chemistry Core Lab made the MALDI-TOF MS instrument available.
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FOOTNOTES |
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* This study was supported by the National Institutes of Health (to C. M. W.) and the Wellcome Trust (to H. R. M., and A. D.).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: University of Florida College of Medicine, 1600 SW Archer Rd., Gainesville, FL 32610-0235. Tel.: 352-392-3329; Fax: 352-392-3305; E-mail: westcm{at}college.med.ufl.edu.
1 The abbreviations used are: Q-TOF MS, quadrupole time-of-flight mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption time-of-flight mass spectrometry; Fuc, L-fucose; Gal, D-galactose; GlcNAc, N-acetyl-D-glucosamine; PCR, polymerase chain reaction; RP-HPLC, reversed phase-high pressure liquid chromatography; GC/MS, gas chromatography mass spectrometry; cFTase, cytosolic fucosyltransferase; TMS, trimethylsilyl.
2 P. Teng-umnuay and C. M. West, unpublished data.
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REFERENCES |
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