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INTRODUCTION |
Insulin-like growth factors
(IGF)1 I and II are peptide
hormones that regulate the differentiation and proliferation of a large number of cell types and also have a role in glucose homeostasis (1).
At least 75% of the total circulating IGFs are carried in 130-150-kDa
ternary complexes containing IGF-binding protein-3 (IGFBP-3) (2) and an
85-kDa glycoprotein, the acid-labile subunit (ALS) (3). Recently,
IGFBP-5 was also found to form a similar ternary complex with the IGFs
and ALS in serum (4). It is thought that the size of the ternary
complex restricts the passage of IGFs to target cells, while free
IGFs, or IGFs in binary complexes with IGFBPs, can cross the
capillary endothelial barrier. Therefore, ALS regulates the
hypoglycemic and mitogenic potential of the circulating IGFs via the
formation of the ternary complexes. Furthermore, ALS plays a vital role
in maintaining a circulating store of the IGFs, IGFBP-3, and possibly
IGFBP-5, by significantly increasing their serum half-lives (5, 6).
Despite the importance of the ternary complexes in regulating serum IGF
bioactivity, nothing is known about the structural aspects of ALS that
enable it to interact with IGFBPs. There are two features of ALS
structure that may play a part. First, the protein backbone of ALS is
made up of repeating blocks each containing 24 amino acids, of which 6 are typically leucine residues. This places ALS in the leucine-rich
repeat superfamily of proteins (7), all of which are involved in
protein-protein interactions (8). Second, serum ALS is heavily and
heterogeneously glycosylated with N-linked glycan chains
(3), and glycosylation is known to influence the interactions of many proteins.
Electrophoretic studies have shown that human ALS circulates as two
glycoforms. Serum-purified ALS displays a characteristic doublet on
SDS-PAGE at 84-86 kDa, which is reduced to a single band of less than
70 kDa after enzymatic removal of the N-linked sugars (3).
There are seven consensus N-linked sugar attachment sites
within the amino acid sequence of ALS that are conserved between
primate and rodent (7, 9-11). One site occurs almost in the center of
the sequence, and a cluster of three sites is found toward each
terminus. Between six and seven bands are observed upon partial
deglycosylation of ALS derived from serum, suggesting that multiple
sites are used (12).
Although there are no studies that directly identify physical
features of ALS involved in ternary complex formation, there is
evidence that charge-charge interaction exists between ALS and the
IGF·IGFBP-3 binary complex. Polyanions, polycations, and increasing
ionic strength all decrease the affinity of ALS for IGFBP-3 (13, 14).
Recently we have shown that the removal of basic residues in the
carboxyl-terminal region of IGFBP-3 decreased its affinity for ALS by
90%, indicating the importance of positive charge in this region (15).
From these observations, it is likely that negative charges on ALS may
be involved in the interaction. At physiological pH, ALS binds tightly
to weak anion exchange columns, indicating that it has a net negative
charge (3).
Since carbohydrates are a potential source of negative charge in
glycoproteins, as well as being involved in protein interactions, we
have investigated whether N-linked sugars on ALS play a role in the formation of ternary complexes between IGFs, IGFBP-3, and ALS.
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EXPERIMENTAL PROCEDURES |
Reagents
Preparations of natural human ALS, human IGFBP-3, and rabbit
antiserum against IGFBP-3 were similar to those used in previous studies (3, 16). ALS was radioiodinated and purified by ion-exchange chromatography as described previously (17). IGF-I (Genentech, San
Francisco, CA) was iodinated and cross-linked to IGFBP-3 as in previous
studies (18). Restriction enzymes and materials for site-directed
mutagenesis were from Promega Corp. (Madison, WI). Bovine serum albumin
(BSA; radioimmunoassay grade, fraction V),
-globulin, hexadimethrine
bromide (Polybrene), dexamethasone, hypoxanthine, xanthine, thymidine,
and mycophenolic acid were purchased from Sigma. Aminopterin was
obtained from Life Technologies Inc.. Nucleoside-free
-modified
Eagle's medium (
-MEM) and fetal calf serum were from Cytosystems
(New South Wales, Australia). Centricon 30 microconcentrators were
obtained from Amicon (Beverly, MA). n-Octyl glucoside,
O-glycosidase
(endo-
-N-acetylgalactosaminidase), and
peptide-N-glycosidase F (PNGase F;
peptide-N4-(acetyl-
-glucosaminyl)-asparagine
amidase) were obtained from Boehringer Mannheim.
N-Acetylneuraminidase III (NANase III) was from Glyko
(Novato, CA). Endo-
-N-acetylglucosaminidase (Endo F
containing <0.1% PNGase F; catalog no. 324706),
2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), and the
sialic acid-specific lectin from Tritichomonas mobilensis
were purchased from Calbiochem (La Jolla, CA).
Enzymatic Deglycosylation and Desialylation of ALS
Characterization of ALS Glycosylation--
PNGase F (5 units)
and NANase III (25 milliunits) were used to remove N-linked
sugars and sialic acids, respectively. Reactions contained
[125I]ALS (4 × 105 cpm, ~40 ng), 50 mM sodium phosphate buffer (pH 6.5), and 0.1% (w/v)
n-octyl glucoside, and were incubated at 37 °C for
16 h. Identical reactions were set up without enzyme as controls.
To investigate O-glycosylation, [125I]ALS
(~40 ng) was treated at 37 °C with 5 units of PNGase F with the
addition of 12.5 milliunits of NANase III after 8 h, then 2 milliunits of O-glycosidase 3 h later. The mixture was
subsequently incubated for an additional 12-16 h at 37 °C.
Enzymatic Deglycosylation for IGFBP-3 Binding Studies--
A
series of increasingly deglycosylated [125I]ALS
preparations was generated by incubation with 2.3, 11.7, 29.3, and 58.6 milliunits of Endo F/ng of [125I]ALS in 50 mM
sodium phosphate buffer, pH 6.5, containing 0.0005% (w/v)
n-octyl glucoside, for 16 h at 37 °C. For complete
deglycosylation, [125I]ALS was treated with 640 milliunits of Endo F/ng of [125I]ALS in 0.0075% (w/v)
n-octyl glucoside.
Desialylated [125I]ALS was prepared using 0.035 milliunits of NANase III/ng of [125I]ALS in 50 mM sodium phosphate buffer (pH 6.5) at 37 °C for 16 h. In experiments where DANA was used, it was added at 1 nmol/milliunit NANase III to specifically inhibit the sialidase activity of NANase III.
Site-directed Mutagenesis of ALS--
The ALS cDNA was
generated by reverse transcriptase-polymerase chain reaction from
normal human liver total RNA as described previously (10). This
cDNA was inserted into the BamHI/XbaI site of
pSELECT (Promega Corp.) for mutagenesis according to the manufacturer's recommended protocol. Mutagenic deoxyoligonucleotides were used to generate seven different ALS cDNAs, each containing Asn to Ala codon substitutions at one of the seven consensus
N-glycan linkage sites (Asn37
Ala,
5'-CTGCAGCTCCAGGgcCCTCACGCGCCTG-3'; Asn58
Ala,
5'-GCTGGACGGCAACgcCCTCTCGTCCGTC-3'; Asn69
Ala,
5'-GGCAGCCTTCCAGgcCCTCTCCAGCCTG-3'; Asn341
Ala,
5'-CGTGGCGGTCATGgcCCTCTCTGGGAAC-3'; Asn448
Ala,
5'-CCTCAGCCTCAGGgcCAACTCACTGCGG-3'; Asn527
Ala,
5'-CTTCGCCCTGCAGgcCCCCAGTGCTGTG-3'; Asn553
Ala,
5'-CCCGCGTACACCTACAACgcCATCACCTG-3'; substituted nucleotides are
shown in lowercase). Oligonucleotides were synthesized using an Oligo
1000 DNA Synthesizer (Beckman, Palo Alto, CA). The mutations were
confirmed by sequencing (T7 sequencing kit; Amersham Pharmacia Biotech), and then the cDNAs were excised from pSELECT and inserted into the NheI/SalI site of pMSG (Amersham
Pharmacia Biotech), an expression vector that contains the
constitutively active and glucocorticoid-inducible murine mammary tumor
virus promoter.
Cell Culture and Transfections--
Chinese hamster ovary (CHO)
cells were transfected with either wild-type or mutant ALS expression
constructs, or pMSG alone using the Polybrene/Me2SO
technique (19). pMSG contains the guanine phosphoribosyltransferase
gene, which confers resistance to mycophenolic acid. Stable
transfectants were selected for 3 weeks in
-MEM supplemented with
10% fetal calf serum, 25 µg/ml mycophenolic acid, 2 µg/ml
aminopterin, 250 µg/ml xanthine, 15 µg/ml hypoxanthine, and 10 µg/ml thymidine. Some ALS transfectants were cultured from single
foci to produce clonal lines. Flasks of stably transfected cells were
grown to confluence in
-MEM supplemented with 10% fetal calf serum,
then the medium was changed to
-MEM supplemented with 10 µM dexamethasone. After 3-4 days, the conditioned medium
was collected and cell debris was removed by centrifugation.
Supernatants were then concentrated and equilibrated into 50 mM sodium phosphate buffer (pH 6.5) using Centricon 30 microconcentrators. Conditioned media from CHO cells transfected with
pMSG alone were concentrated to the same extent as media containing the
lowest concentration of mutant ALS, and used as controls.
Electrophoretic Analyses
SDS-PAGE Analysis--
Radioiodinated ALS (5,000 cpm, ~0.5 ng)
in Laemmli buffer was loaded, without heat treatment, onto 7.5% Ready
gels (Bio-Rad) and electrophoretically separated under nonreducing
conditions. The gels were then dried and exposed to Hyperfilm MP
(Amersham, Bucks, UK) overnight at
80 °C.
Isoelectric Focusing of ALS--
Preparations of
[125I]ALS (10,000 cpm, ~1 ng), untreated or treated
with NANase III, were added to sample buffer containing 4% (w/v) CHAPS
(BDH Ltd, Poole, UK), 100 mM Tris-HCl, pH 7.2, and 2%
(v/v) ampholytes (Pharmalyte 3-10, Amersham Pharmacia Biotech). Samples were loaded onto the alkaline end of an immobilized pH gradient
strip (pH 3-10, Amersham Pharmacia Biotech) that had been rehydrated
overnight in 0.5% (w/v) CHAPS, 10 mM dithiothreitol, 6 M urea, and 2% (v/v) Pharmalyte 3-10. Isoelectric focusing
was performed in a Multiphor II electrophoresis unit (Amersham
Pharmacia Biotech). A broad pI isoelectric focusing calibration kit (pH 3-10; Amersham Pharmacia Biotech) was run in parallel with the ALS
samples. The theoretical pI for the human ALS protein backbone was
calculated using the program ISOELECTRIC from the Genetics Computer
Group, Inc. (Madison, WI).
Binding Assays
Gel Filtration Studies with Mutated ALS--
Size fractionation
chromatography was used to determine whether the seven site-directed
mutant recombinant ALS species lacking individual consensus
N-glycan linkage sites were able to form ternary complexes
(18). Briefly, cross-linked [125I]IGF-I·IGFBP-3 (1 × 105 cpm) was incubated for 30 min at 25 °C with
conditioned medium containing 150 ng of mutant ALS equilibrated in 50 mM sodium phosphate buffer (pH 6.5) containing 1% (w/v)
BSA, in a total volume of 200 µl. The mixture was then injected into
a Superose-12 column (Amersham Pharmacia Biotech), and 0.5-ml fractions
of eluate were collected at a flow rate of 1 ml/min. The degree of
conversion from binary to ternary complex was evaluated by the shift of
radioactivity from fractions corresponding to 50 kDa to those
corresponding to 140 kDa (18).
Lectin Solution Binding Assay--
A lectin solution binding
assay was used to indicate the presence or absence of sialic acids on
[125I]ALS after enzymatic desialylation with NANase III
and deglycosylation with PNGase F as described above. Identical
reactions were set up without enzymes as controls. The assay was
modified from that of Abidi et al. (20). Briefly,
approximately 35,000 cpm [125I]ALS (~3.5 ng), either
treated with enzyme or untreated control, was incubated for 1 h
with 2 µg of the sialic acid-specific lectin from T. mobilensis in 50 mM sodium phosphate buffer with
0.01% (w/v) BSA, pH 6.5 at 22 °C (final volume 100 µl).
[125I]ALS complexed to the lectin was then precipitated
using
-globulin (35 µl) and 6% polyethylene glycol (1 ml) for 10 min at 4 °C. Both BSA and
-globulin were acid-hydrolyzed and
dialyzed against water to remove contaminating sialic acids (21). The
tubes were then spun at 3500 rpm in a swing bucket centrifuge for 10 min at 22 °C. The radioactive pellet in each tube was measured as a
percentage of the total radioactivity added and these data were used to
generate histograms. Nonspecific binding was determined to be the
radioactivity measured when no lectin was added to the tube during the
1-h incubation. Control and desialylated forms of
[125I]ALS gave nonspecific binding ranging from 10% to
14% of total, and the PNGase F deglycosylated forms of
[125I]ALS gave approximately 28% of total.
Solution Binding Assay and Scatchard Analysis--
Solution
binding assays were carried out as described previously (16). Briefly,
10,000 cpm [125I]ALS, either treated with enzyme or
untreated control, was incubated for 2 h with 10 ng of IGF-I or
-II and a range from 0 to 10 ng of IGFBP-3 in 50 mM sodium
phosphate buffer, pH 6.5, at 22 °C (final volume 0.3 ml). ALS
complexed to IGFBP-3 was then precipitated using IGFBP-3 antiserum. The
radioactivity in each tube was measured as a percentage of the total
radioactivity added, and these data were used to generate binding
curves. Endo F (58.3 milliunits/ng ALS) was added to a control
untreated [125I]ALS preparation during the 2-h incubation
to ensure that the presence of the enzyme did not adversely affect
complex formation. Nonspecific binding was calculated as the percentage
of radioactivity present after precipitation when there was no IGFBP-3
in the reaction mixture. For the Endo F deglycosylation experiments,
nonspecific binding ranged from 3% to 18% of total for the partially
deglycosylated forms and was approximately 30% of total for the fully
deglycosylated forms. For the the NANase III binding curves,
nonspecific binding was between 3% and 8% of the total. Scatchard
analysis was carried out as described previously (16), except that
IGF-I, IGF-II, and IGFBP-3 were held constant at 1 ng/0.3 ml. ALS was
added over the range of 0-200 ng/0.3 ml.
Statistical Analyses--
Binding curve data were analyzed by
repeated measures analysis of variance, followed by Fisher's protected
least significant difference test, using Statview 4.02 (Abacus Concepts
Inc., Berkeley, CA). The value was considered significant if the
p value was less than 0.05.
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RESULTS |
Glycosidase Characterization of ALS Carbohydrates--
ALS is
estimated to have ~20 kDa of N-linked carbohydrate, but
the possible presence of O-linked sugars has not been
investigated. To address this, [125I]ALS was treated with
NANase III, which removes sialic acid, and O-glycosidase,
which hydrolyzes some of the common core sugars of O-linked
glycans. SDS-PAGE analysis was then used to identify shifts in the
apparent size of treated ALS. Untreated [125I]ALS (Fig.
1, lane 1) appears
as a single band of approximately 85 kDa. The diffuse nature of the
band may be explained by poor resolution of the 84-86-kDa doublet. The
apparent size of ALS after NANase III treatment (lane
2) was decreased compared with untreated ALS. The
approximate size difference is 2-3 kDa, consistent with the removal of
5-15 sialic acid moieties. ALS was also treated with PNGase F, an
effective amidase that cleaves all types of N-linked sugars,
to show the previously reported size shift to approximately 68-70 kDa
(lane 3). This apparent molecular mass is very
close to the predicted value of 66 kDa for the amino acid backbone of
ALS. However, there is no difference in apparent size between ALS
treated with PNGase F alone (lane 3) and ALS
treated with PNGase F, NANase III and O-glycosidase
(lane 4). These results suggest that ALS carries
sialic acids that are predominantly or entirely attached to the
N-linked sugar chains.

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Fig. 1.
SDS-PAGE analysis of ALS after treatment with
various glycosidases. Preparations of [125I]ALS were
incubated with the glycosidases indicated (see "Experimental
Procedures") and electrophoretically separated by nonreducing
SDS-PAGE. Radiolabeled proteins were detected by autoradiography. The
molecular mass markers are shown to the right.
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However, it is possible that, following O-glycosidase
treatment, electrophoresis did not resolve the small shift in size
predicted if O-linked sugars are a minor component of ALS
carbohydrate. To investigate this further, we used a lectin binding
assay (Fig. 2) to determine whether the
sialic acids present on ALS could be accounted for by the
N-linked sugars alone. A sialic acid-specific lectin derived
from T. mobilensis was used to precipitate
[125I]ALS, which was either untreated or had been
desialylated with NANase III or deglycosylated with PNGase F. Using
this assay, we found that desialylation and deglycosylation caused
96 ± 4% and 88 ± 5% loss of binding, respectively. Since
there was no significant difference between these values
(p = 0.1, t test), we conclude that all of
the sialic acids removed by NANase III treatment are derived from
N-linked sugars on ALS.

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Fig. 2.
The sialic acids of ALS removed by NANase III
treatment are on the N-linked sugars of ALS.
Preparations of [125I]ALS in 50 mM phosphate
buffer, NANase buffer without or with NANase III and PNGase F buffer
without or with PNGase F were incubated overnight at 37 °C. The
treated [125I]ALS preparations were then assayed for
binding to a sialic acid-specific lectin. All data were corrected for
nonspecific binding. The data represent means ± S.E. of three
separate experiments.
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Charge Contribution of the Sialic Acids to ALS--
We used
isolectric focusing to establish the net negative charge that the
sialic acids would contribute to ALS. Untreated [125I]ALS
and NANase III-treated [125I]ALS were separated by charge
on immobilized pH gradient strips and visualized by autoradiography
(Fig. 3). Untreated ALS was found to have
at least six distinctly charged isoforms in the pI range of 4.5-5.2.
Although the NANase III-treated ALS was less well resolved than the
control, it is clear that the pI of the desialylated
[125I]ALS had shifted, as expected, toward a more neutral
pI value of approximately 5.5-7.0. Furthermore, the distinct series of bands observed in the control disappeared after NANase III treatment. Therefore, the six distinct isoforms of ALS are consistent with a
series of differentially sialylated ALS molecules.

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Fig. 3.
Isoelectric focusing profiles of native and
NANase III-treated ALS. Untreated and NANase III-treated
[125I]ALS were loaded at the alkaline ends (~pH 9) of
immobilized pH gradient strips (pH 3-10) and focused
electrophoretically (see "Experimental Procedures"). The relative
positions of pI standards are indicated. The theoretical pI of the core
ALS protein is indicated by an arrow.
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Complete Deglycosylation of ALS Abolishes Ternary Complex
Formation--
In order to identify a potential biological role for
ALS glycosylation, we investigated the effect that N-linked
deglycosylation of ALS has on the formation of ternary complexes
between the IGFs, IGFBP-3, and ALS. Enzymatic methods were used to
deglycosylate serum-derived ALS. After radiolabeling,
[125I]ALS was treated with Endo F, an endoglycosidase
mixture able to remove most types of N-linked sugars. The
preparation of Endo-F used contained only trace levels of the
asparagine amidase PNGase F. This was to limit the potentially
confounding effects of converting the carbohydrate-anchoring asparagine
to an aspartic acid (22, 23). SDS-PAGE analysis and autoradiography
were used to monitor the degree of deglycosylation attained (Fig.
4, A and C), and the resulting preparations were used in a solution binding assay to
measure their ternary complex forming ability (Fig. 4, B and D). Fig. 4A depicts the SDS-PAGE analysis of
[125I]ALS treated under conditions that fully removed
N-linked sugars. The binding curves (Fig. 4B)
reveal that there is no specific binding of either the IGF-I·IGFBP-3
or the IGF-II·IGFBP-3 binary complex with the fully deglycosylated
ALS preparation. Therefore, the complete removal of N-linked
sugars from ALS by Endo F abolishes ternary complex formation.

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Fig. 4.
Partial or complete enzymatic deglycosylation
reduces or abolishes, respectively, the ability of ALS to bind to
IGFBP-3. Preparations of [125I]ALS were treated with
Endo F for 16 h at 37 °C, subjected to nonreducing SDS-PAGE
(panels A and C), and assayed for binding to
increasing amounts of IGFBP-3 in the presence of 10 ng of IGF
(panels B and D). Panel A, samples of
untreated and completely deglycosylated [125I]ALS
subjected to SDS-PAGE and then detected using autoradiography.
Panel B, binding curves of untreated (open
symbols) and deglycosylated (closed symbols) ALS to
IGFBP-3 in the presence of either IGF-I (squares) or IGF-II
(circles). Deglycosylation of the [125I]ALS
used in these experiments was confirmed by SDS-PAGE (see panel
A). Data shown are means ± S.E. of three separate
experiments. Panel C, samples of [125I]ALS
partially deglycosylated with increasing concentrations of Endo F,
subjected to SDS-PAGE, then detected by autoradiography. Panel
D, binding of the natural and partially deglycosylated ALS samples
shown in panel C to IGFBP-3·IGF-I complexes ( , ,
, , and represent 0, 2.3, 11.7, 29.2, and 58.3 milliunits of
Endo F/ng of ALS, respectively). Similar binding curves were seen in
two independent experiments.
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We also generated a series of partially deglycosylated
[125I]ALS preparations (Fig. 4C). As seen in
the binding curves depicted in Fig. 4D, the most effectively
deglycosylated preparations were the least able to form complexes with
IGF-I and IGFBP-3, whereas the preparation with almost fully intact
N-glycosylation was virtually indistinguishable from the
control. Therefore, although the complete removal of
N-linked sugars from ALS abolishes ternary complex formation, ALS with some intact N-linked sugars is able to
form the complex.
No N-Linked Sugar Is Solely Responsible for ALS Binding
Activity--
The binding ability of a protein can be substantially
altered by the removal of a single N-linked carbohydrated
chain (24). Since we demonstrated that the N-linked sugars
on ALS have a role in ternary complex formation, we used site-directed
mutagenesis to investigate whether any single glycan chain was solely
responsible for the binding of ALS to the IGF and IGFBP-3 complex. The
primary sequence of ALS has seven consensus NXS/T sites for
N-linked sugar attachment. Therefore, we constructed a
series of seven mutant ALS cDNAs. The mutations were at
Asn37
Ala, Asn58
Ala, Asn69
Ala, Asn341
Ala, Asn448
Ala,
Asn527
Ala and Asn553
Ala. The mutant
ALS cDNAs were then transfected into CHO cells, and the proteins
were harvested. All transfections resulted in conditioned medium
containing measurable amounts of immunoreactive ALS, as determined by
radioimmunoassay (17). From this, we concluded that no single
N-linked sugar is an absolute requirement for secretion of
ALS by CHO cells.
The conditioned media from the transfectants were then used to
determine the ternary complex forming ability of the various glycosylation mutant ALS proteins. Ternary complex formation was evaluated by size shift of a cross-linked
[125I]IGF-I·IGFBP-3 complex on a Superose-12 gel
permeation column (Fig. 5). The peak
radioactivity found in fraction 26 with the pMSG control (Fig.
5A) is consistent with the size of the IGF-I·IGFBP-3 binary complex (18). In contrast, media from wild-type ALS transfected CHO cells caused a clear shift in the peak fraction of radioactivity from fraction 26 to fraction 23 (Fig. 5A). Similar
chromatography profiles were obtained for all the mutant ALS forms and
are depicted in Fig. 5B, except Asn448
Ala,
which gave similar results in a separate assay (data not shown). In
each case, the shift in peak radioactivity from fraction 26 to fraction
23 is consistent with the formation of ternary complexes. Therefore, we
conclude that all of the mutant ALS proteins are able to bind to the
IGF-I·IGFBP-3 complex. Hence, no single N-linked sugar of
ALS can solely account for the loss of activity observed in the
enzymatic deglycosylation experiments.

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Fig. 5.
ALS mutants lacking individual glycan
attachment sites bind to IGFBP-3. Panel A,
cross-linked [125I]IGF-I·IGFBP-3 was incubated with
media conditioned by CHO cells transfected with pMSG ( ) or 150 ng of
recombinant wild type ALS ( ) in a volume of 200 µl and then
analyzed by size fractionation on a Superose-12 column. The
open and closed arrows represent
~140- and ~50-kDa complexes, respectively. Panel B, in a
similar series of experiments cross-linked
[125I]IGF-I·IGFBP-3 was incubated with media containing
150 ng of recombinant mutant ALS species lacking specific glycan
attachment sites ( , Asn37 Ala; ,
Asn58 Ala; , Asn69 Ala; ,
Asn341 Ala; , Asn527 Ala; ,
Asn553 Ala), and resulting complexes were analyzed on a
Superose-12 column. In a separate experiment, recombinant ALS mutant
Asn448 Ala displayed a similar increase in size when
incubated with [125I]IGF-I·IGFBP-3.
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NANase III-treated ALS Has a Reduced Affinity for
IGFBP-3--
Having established that ALS glycosylation has a role in
ternary complex formation, we investigated whether negatively charged sialic acid moieties may be specifically involved in ALS binding to the
IGF·IGFBP-3 complex. Binding curves for the interaction of NANase
III-treated ALS and untreated ALS with IGFBP-3 are depicted in Fig.
6. In the presence of IGF-I,
desialylation of ALS significantly shifted the binding curve to the
right (p = 0.02) (Fig. 6B), indicative of a
decrease in ALS binding. A similar result was obtained in the presence
of IGF-II (p = 0.005) (Fig. 6C). Therefore,
ternary complex formation in the presence of either IGF-I or IGF-II is reduced after enzymatic desialylation of ALS. One of the desialylated preparations used in this study was also used in the IEF depicted in
Fig. 3, demonstrating an increase in pI compared with control [125I]ALS and demonstrating that the preparation was
fully desialylated under the conditions used.

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Fig. 6.
NANase III-treated ALS has a reduced ability
to bind IGFBP-3. Preparations of [125I]ALS were
treated with or without NANase III overnight at 37 °C and then
subjected to nonreducing SDS-PAGE; a representative autoradiograph is
shown in panel A. The untreated ( ) and NANase III-treated
( ) [125I]ALS were then assayed for binding to
increasing amounts of IGFBP-3 in the presence of 10 ng of IGF-I
(panel B) and IGF-II (panel C). Desialylation of
the [125I]ALS used in these experiments was confirmed by
SDS-PAGE (see panel A). The binding curves represent
means ± S.E. of three separate experiments.
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The specificity of NANase III treatment was tested by blocking its
sialidase activity with the sialic acid analog DANA. ALS treated with
NANase III in the presence of DANA showed IGFBP-3 binding activity that
was not significantly different from the untreated protein (Fig.
7). This suggested that NANase III
specifically removed the sialic acid moieties from ALS and that these
sialic acid moieties were necessary for normal ternary complex
formation.

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Fig. 7.
Binding of ALS to IGFBP-3 is specifically
dependent on sialylation. Preparations of [125I]ALS
either treated with NANase III ( ) or with NANase III in the presence
of the sialic acid analog DANA (1 nmol/milliunit NANase III, ) or
with no treatment ( ) were incubated for up to 16 h at 37 °C
(see "Experimental Procedures"). These preparations were then
assayed for binding to increasing amounts of IGFBP-3 in the presence of
10 ng of IGF-I. All complex formation curves were corrected for
nonspecific binding and then expressed as the percentage of maximum
binding of the control (untreated [125I]ALS in the
presence of 10 ng of IGFBP-3 and 10 ng of IGF-I).
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Finally, Scatchard analysis was used to determine the difference in
affinities of NANase III-treated ALS and untreated ALS for
IGF-I·IGFBP-3 and IGF-II·IGFBP-3 complexes. Representative Scatchard plots for IGF-I ternary complex formation are shown in Fig.
8, indicating a decrease in affinity of
ALS for IGFBP-3 after desialylation. Representative association
constants derived from two experiments are shown in Table
I. These experiments show that
desialylated ALS has a 50-80% reduction in affinity for IGFBP-3 in
the presence of IGF-I or IGF-II compared with normally sialylated
ALS.

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Fig. 8.
Reduced affinity of desialylated ALS for
ternary complex formation. Representative Scatchard analyses
comparing the affinities of untreated (panel A) and NANase
III-treated (panel B) ALS for 1 ng of IGFBP-3 in the
presence of 10 ng of IGF-I.
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Table I
Summary of affinities of untreated and NANase III-treated ALS for
IGF·IGFBP-3 complexes
The affinities of untreated and desialylated ALS for both
IGF-I·IGFBP-3 and IGF-II·IGFBP-3 complexes were
determined by Scatchard analyses. The results are derived from two
separate experiments.
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DISCUSSION |
The interaction of ALS with IGF·IGFBP-3 complexes in the serum
is believed to regulate both the function and the stability of the
bound IGFs and IGFBP-3. Previous reports indicate that the affinity of
IGFBP-3 for its ligands may be affected by post-translational modifications such as limited proteolysis (25-27). In this study, we
found that the carbohydrate chains on ALS play an influential role in
determining its interaction with IGFBP-3. Our data therefore suggest
that modifications to ALS as well as IGFBP-3 may be important in
fine-tuning the bioavailability of the IGFs.
In addition to the seven putative N-linked carbohydrate
attachment sites (NXS/T) in the ALS sequence, there are two
putative O-linked glycan sites. The first, at
Ser60, conforms to a mucin-type O-glycosylation
site as predicted by NetOglyc 2.0 (28) and is part of the
N-linked carbohydrate attachment site at Asn58.
The second, FT494PQP, corresponds to the
XTPXP sequence recently described as the minimal
requirement for O-linked carbohydrates (29) and lies adjacent to the putative N-linked glycosylation site,
Asn448. Treatment of ALS with O-glycosidase,
which hydrolyzes Gal
(1-3)GalNAc, a common core structure of many
O-linked glycans (30), had no effect on the molecular mass
of ALS as judged by SDS-PAGE analysis. While it is possible that fucose
residues may have interfered with the enzyme, or that a shift in size
was below the level of detection, the putative O-linked
sites do not appear to be occupied by sugar side chains with the common
core structure described above. Furthermore, a lectin binding assay
that was specific for sialic acids, a common residue on
O-linked carbohydrates, also failed to provide evidence for
O-linked sugars on ALS.
Therefore, we focused on the N-linked carbohydrates of ALS
and their role in ternary complex formation. Enzymatic removal of the
N-linked sugars from ALS decreased its ability to form the
complex with IGFBP-3 in a manner that was related to the level of
deglycosylation. Intermediate levels of ALS deglycosylation, probably
involving the loss of more than a single sugar chain, measurably
disrupted ternary complex formation, whereas complete removal of the
N-linked glycans abolished complex formation. However, when
site-directed mutagenesis was used to mutate each of the N-linked attachment sites individually, no single glycan
appeared to have a major impact on ALS binding activity. Therefore, the carbohydrates on ALS clearly influence the affinity that ALS has for
the IGF·IGFBP-3 complex, although this influence relies on a number
of N-linked sugars rather than any single chain. However, it
is not clear whether deglycosylated ALS is less able to form the
ternary complex because of conformational changes in ALS induced by
removal of the glycans or disruption of interactions between the ALS
carbohydrates and the other two proteins.
Sialic acids are common anionic residues that can be attached to both
N- and O-linked sugars. They can contribute
significant charge to glycoproteins as many sialic acids can be
attached to one highly branched N-linked sugar. For example,
the acute-phase protein
1-acid glycoprotein has a pI of
2-3 mostly due to the large number of sialic acids attached to its
highly branched complex N-linked sugars (31). The calculated
pI of ALS, based on its amino acid sequence, is 6.56; however, we
observed six discrete isoforms of pI 4.6-5.3 by isoelectric focusing.
After NANase III treatment, the pI of ALS increased to between 5.5 and
7, close to the predicted value for the amino acid backbone. The
contribution of negative charge by the sialic acid may explain, in
part, the high affinity with which ALS binds to weak anion exchange
columns used in ALS purification (3). Given that the sialic acid
contributes significantly to the negative charge on ALS and that the
interactions within the ternary complex are dependent on charge, it
could be predicted that the sialic acid on ALS would affect the
formation of the complex. Indeed, ALS treated with NANase III to remove sialic acid displayed a 50-80% decrease in affinity for the IGF-I and
IGF-II binary complexes compared with that of untreated ALS. However,
it is noteworthy that desialylation only lowered the affinity of ALS
for the IGF·IGFBP-3 complex unlike deglycosylation, which abolished
complex formation. This suggests that the effects of ALS glycosylation
on IGFBP-3 binding are not entirely due to the negative charges
imparted by sialic acid.
The results from the N-linked glycan studies imply that a
number of N-linked sugars are required to act in concert to
enable ALS to interact with IGFBP-3 and the IGFs, rather than a
specific carbohydrate chain being solely responsible. The placement of the glycans within the tertiary structure of ALS may shed light on this
finding. In unpublished
studies,2 we have modeled the
central leucine-rich repeat region of ALS on the only published crystal
structure of a leucine-rich repeat protein, the porcine ribonuclease
inhibitor (32). If the modeled structure is a true representation of
ALS, then six out of the seven potential N-linked sugar
attachment sites lie very close to each other, suggesting a possible
clustering of carbohydrate chains. Therefore, the loss of any single
N-linked glycan may be compensated by the potentially large
number of other carbohydrates in the vicinity. This model also has
implications with regard to the sialic acid moieties that we
demonstrated to exist on ALS. A lectin solution binding assay suggested
that all the sialic acids on ALS are attached to the
N-linked sugars. These sialic acid moieties might therefore
result in a region of negative charge where the N-linked
sugars are concentrated.
The binding affinity of ALS to the IGF·IGFBP-3 complex is relatively
weak, 1-2 orders of magnitude less than the affinity of IGFBP-3 for
either of the IGFs (13), and in this sense ALS binding is the limiting
step in ternary complex formation. This suggests that modulation of ALS
affinity might directly influence complex formation, and thus the
bioavailability of IGFs. Isoelectric focusing indicates that ALS
purified from normal serum already exists as a number of differently
sialylated isoforms. If the degree of ALS sialylation is subject to
physiological regulation, as described for other proteins (31), the
potential exists for modulation of formation or stability of the
ternary complexes. This might occur in addition to the previously
described modulation of the circulating concentration of the ALS
protein itself (33) through cytokine suppression (34) or in patients
who are critically ill (35) or have hepatic cirrhosis (36).
In summary, we have found that the N-linked carbohydrates on
ALS are a requirement for the formation of complexes with IGFBP-3 and
IGFs. We have also shown that the removal of sialic acids from ALS
significantly reduces the affinity of ALS for binding to IGFBP-3. Since
the glycosylation of secreted proteins are often modified in certain
physiological and pathological states, the modification of ALS
glycosylation has the potential to be an important factor in the
regulation of IGF access to the tissues.