From the
We have characterized a soluble form of the insulin-like growth
factor II/mannose 6-phosphate receptor (sIGF-II/MPR) and bound ligands
from bovine serum. Fetal serum contained 2-8 mg/liter
sIGF-II/MPR. Affinity-purified receptor isolated by adsorption to
phosphomannan-agarose and elution with mannose 6-phosphate contained
nearly stoichiometric amounts of bound 7.5-kDa IGF-II. In addition, at
least 12 distinct 12-20-kDa proteins immunologically related to
IGF-II also copurified with receptor. Receptor was separated from its
associated ligands by acidification and gel filtration chromatography.
Sequence analysis revealed that the 12-20-kDa proteins have the
same amino termini as mature 7.5-kDa IGF-II. Protease and glycosidase
treatments revealed that the different high molecular weight IGF-II
species contain an identical COOH-terminal extension that is
differentially glycosylated with O-linked sugars. Radiolabeled
tracer experiments demonstrated that the sIGF-II/MPR carries
The insulin-like growth factor II/mannose 6-phosphate receptor
(IGF-II/MPR)
The extracytoplasmic ligand-binding region of the receptor
contains 15 repeating units that have an average length of 147 amino
acids and are 16-38% identical (Lobel et al., 1988). The
receptor contains one IGF-II and two Man-6-P binding sites (Tong et
al., 1989), and it is likely that each binding site is formed by a
different repeating unit (Dahms et al., 1993, 1994; Garmroudi
and MacDonald, 1994). One fascinating question is why the receptor
contains 15 repeating domains. While it is possible that three units
bind ligands and the other 12 are structural components, another
possibility is that the receptor functions in lysosomal targeting of
other ligands not yet identified. One of the purposes of this study was
to search for endogenous non-Man-6-P-containing ligands of the
receptor.
A soluble form of the IGF-II/MPR has been detected in
serum and urine (Kiess et al., 1987b; Gelato et al.,
1988; Causin et al., 1988; Gelato et al., 1989; Li et al., 1991). This protein retains IGF-II and Man-6-P binding
activities and, based on its size, contains nearly the entire
extracytoplasmic domain. Both the membrane-bound and soluble forms of
the receptor are present at high levels during fetal development. In
this study, we isolated and characterized the soluble receptor and its
bound ligands from fetal bovine serum. While we did not detect any new
classes of ligands, we found that in addition to mature 7.5-kDa IGF-II,
the receptor binds at least 12 different high molecular weight forms of
IGF-II. These proteins have apparent molecular weights of
12,000-20,000 and contain a common peptide backbone modified with
different amounts of O-linked sugars. Furthermore, we
demonstrate that the soluble receptor carries a significant fraction of
IGF-II in fetal bovine serum.
Receptor and
non-Man-6-P-containing ligands were isolated using a three-step scheme
(see ``Experimental Procedures''). First, purification on a
Man-6-P affinity resin (phosphomannan-agarose) was used to isolate
receptor and to remove endogenous Man-6-P-containing ligands. Second,
gel filtration chromatography at neutral pH was used to separate
receptor and bound ligands from low molecular weight contaminants. As
the receptor contains two Man-6-P binding sites (Tong et al.,
1989), Man-6-P was included in this step to guard against the unlikely
possibility that some Man-6-P-containing ligands remained associated
with the receptor during affinity purification. Third, gel filtration
chromatography was performed at mildly acidic pH to separate receptor
from ligands. The final step capitalizes on the functional properties
of this recycling receptor, which binds ligands at the near neutral pH
of the Golgi or extracellular compartment and releases ligands into the
acidic endosome.
Initial affinity purification of receptor was quite
efficient: enzyme-linked immunosorbent assay on the starting material
and run through of the phosphomannan column demonstrated that in a
typical preparation, >95% of the receptor adsorbed to the resin. The
material eluted with Man-6-P was concentrated by ultrafiltration and
fractionated by gel filtration chromatography at neutral pH (Fig. 1A, upperpart). SDS-PAGE and
Coomassie staining showed that both the 250-kDa receptor and a 6-kDa
protein eluted near the void volume (Fig. 1A, peakN1; Fig. 1B, Coomassie, pH 7.4),
while silver staining revealed that several additional proteins were
present in the peak (Fig. 1B, silver, pH
7.4, 108 and 120ml). The lower
molecular weight proteins were tightly associated with the receptor,
since an identical staining pattern was observed for the material
eluted from the phosphomannan column, the retentate from the
ultrafiltration step, and the fractions in peak N1. In contrast, no
protein was detected in the ultrafiltrate or later eluting fractions
from the neutral gel filtration columns (data not shown).
A specific
subset of the lower molecular weight species represents ligands that
copurify with the sIGF-II/MPR. After acidification, gel filtration
chromatography resolved peak N1 into three fractions (Fig. 1A, lowerpart). The major peak
eluted near the void volume (peak A1), while two minor peaks eluted in
the included volume (peaks A2 and A3). The centers of peaks A2 and A3
eluted as 18- and 7-kDa globular proteins, respectively. SDS-PAGE and
Coomassie staining showed that peak A1 contained the 250-kDa receptor,
while peak A3 contained the 6-kDa protein (Fig. 1B, Coomassie, pH4.5, 120 and 228ml, respectively). In addition, silver staining revealed
that peak A2 contained some of the 6-kDa protein and a series of other
proteins with apparent molecular weights in the range of
12,000-20,000 (Fig. 1B, silver, pH4.5, 168-216 ml). Note that compared with
peak N1, peak A1 exhibits increased staining of multiple bands
(25-200 kDa). However, this smear probably represents nicked
fragments of the sIGF-II/MPR generated by prolonged acidification. The
important finding is that compared with peak N1, peak A1 exhibits
decreased staining of the 6- and 12-20-kDa bands that are now
present in peaks A3 and A2, respectively. This suggests that these
proteins represent true ligands of the receptor. In contrast, sequence
analysis demonstrated that the 66-kDa band seen in peaks N1, A1, and
some of A2 represents contaminating bovine serum albumin.
In addition to soluble receptor, there are at
least six other IGF binding proteins that have molecular weights
ranging from 25 to 150 kDa (for review, see Lamson et
al.(1991) and Clemmons(1993)). To estimate the fraction of IGF-II
carried by the sIGF-II/MPR, fetal bovine serum was incubated with
iodinated bovine IGF-II and applied to a gel filtration column (White et al., 1982; Arner et al., 1989). Time course
experiments indicated that 24-h incubation at room temperature was
sufficient to approach equilibrium. This occurred in a biphasic
process. At both early and late time points nearly all tracer bound to
serum proteins, with
Several different models could explain the
biphasic binding of tracer to serum proteins. For instance, this may
represent rapid binding of tracer to unoccupied low M
Binding experiments
using different lots of serum indicated that 23-32% of the tracer
was in a peak that eluted identically to sIGF-II/MPR standard (Fig. 9A, shadedregion; ). Furthermore, the fraction of tracer associated with
this peak was proportional to endogenous receptor levels (). Most of this IGF-II binding activity represents
authentic sIGF-II/MPR, since receptor-depleted serum has greatly
diminished activity (Fig. 9B, shadedregion; ). Interestingly, while
enzyme-linked immunosorbent assay () and Western blotting
analysis using polyclonal antisera (data not shown) demonstrated
complete removal of receptor, 4-5% of the IGF-II tracer still
migrated in this region. This binding is specific, since excess
unlabeled rhIGF-II resulted in elution of tracer as the free 7.5-kDa
protein (Fig. 9C). Thus, while another protein makes a
minor contribution to IGF-II binding in this region, these results
clearly show that the endogenous sIGF-II/MPR carries
These studies were initiated to identify ligands that bind to
the IGF-II/MPR at regions distinct from the Man-6-P binding sites. Our
approach was to isolate receptor and bound ligands on a phosphomannan
resin, reasoning that Man-6-P-containing proteins would be displaced
without disrupting interactions at other binding sites. We purified a
soluble form of the receptor from fetal serum, eliminating the need for
detergent solubilization of membrane fractions that could potentially
disrupt receptor-ligand complexes. In addition, we used a buffer that
reflected the total ionic composition of human plasma (Lentner, 1984)
to supply possible cofactors required for ligand binding. Using this
strategy, we successfully isolated both mature 7.5-kDa IGF-II and a
fraction containing at least 12 different higher molecular weight
ligands. Given the complexity of the latter fraction, we used multiple
approaches to determine its composition. Activity blotting with soluble
receptor, affinity and immunoprecipitation experiments using
immobilized receptor, and an anti-IGF-II monoclonal antibody,
respectively, and amino-terminal sequencing indicate that all the
proteins isolated represent high molecular weight forms of IGF-II.
There are several possible reasons why this strategy would not
detect other, yet unidentified, classes of ligands that may bind to the
IGF-II/MPR. For instance, this approach requires that there be no
steric or allosteric interactions that prevent copurification of
receptor and ligand. It has been shown that Man-6-P and IGF-II do not
compete with each other for binding to the receptor, while the
phosphorylated lysosomal enzyme
The sIGF-II/MPR has been detected in human (Causin et al.,
1988), primate (Gelato et al., 1988), rodent (Kiess et
al., 1987b), ovine (Gelato et al., 1989), and bovine sera
(Yang et al., 1991; Li et al., 1991) and in human
urine (Causin et al., 1988). The circulating form is
developmentally regulated, with the highest levels occurring in fetal
serum (Kiess et al., 1987b; Gelato et al., 1988;
Gelato et al., 1989; Li et al., 1991). The
sIGF-II/MPR from different species carries variable amounts of
circulating IGF-II; 3, 20, and 50% of the total circulating IGF-II in
fetal sera from rat (Kiess et al., 1987b), monkey (Gelato et al., 1988), and sheep (Gelato et al., 1989),
respectively, was associated with a high molecular weight protein that
was excluded from Sephadex G-200. Chemical cross-linking studies with
radiolabeled IGF-II indicated that the receptor was the major carrier
in these fractions. In addition, receptor purified on
pentamannosylphosphate-agarose from fetal bovine serum contained
substantial amounts of bound IGF-II, since amino-terminal sequencing
yielded equivalent levels of both proteins (Li et al., 1991).
Our results confirm and extend these findings, demonstrating that
An important finding of this study is that at least 12
different high molecular weight forms of IGF-II are associated with the
circulating fetal sIGF-II/MPR. Different variant and high molecular
weight IGF-IIs have been reported previously (for review, see
Humbel(1990) and Rechler(1991); also see Hudgins et
al.(1992)). Human IGF-II variants that are substituted at Ser-29
and Ser-33 with RLPG and CGD, respectively, with or without
COOH-terminal extensions, have been identified (Zumstein et
al., 1985; Hampton et al., 1989). Proteolysis experiments (Fig. 6) indicate that our high M
The existence of the sIGF-II/MPR and bound ligands
in serum may have important experimental and biological implications.
First, fetal serum is frequently used in mammalian cell culture
experiments. While it is now appreciated that IGF-II added to
serum-containing media can be buffered by binding proteins, the added
IGF-II could also displace high M
Two
independent sample dilutions were assayed for sIGF-II/MPR by
enzyme-linked immunosorbent assay as described by Chen et al. (1993). The n values represent the number of different
serum lots or samples from different animals (other fluids).
We thank Henry Lackland and Stanley Stein of the
Center for Advanced Biotechnology and Medicine Protein Chemistry
Laboratory for protein sequencing and amino acid analyses.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of
the IGF-II in fetal bovine serum. These results support a significant
role for sIGF-II/MPR in the transport of circulating IGF-II isoforms
during development.
(
)is an integral membrane protein
that binds two distinct classes of ligands: mannose 6-phosphate
(Man-6-P) containing proteins and insulin like-growth factor II
(IGF-II) (for review, see Kornfeld(1992) and Hoflack and Lobel (1993)).
The Man-6-P modification on N-linked oligosaccharides of
multiple newly synthesized lysosomal enzymes allows them to bind to
intracellular IGF-II/MPRs. The complexes then exit the Golgi and travel
to an acidic prelysosomal compartment (endosome) where the low pH
promotes dissociation of ligand from receptor. Free receptors recycle
back to the Golgi to repeat the process or to the cell surface where
they mediate the endocytosis and lysosomal targeting of extracellular
phosphorylated lysosomal enzymes. The function of the receptor in
binding IGF-II is controversial. Mature IGF-II is a nonphosphorylated,
nonglycosylated 67-amino acid protein related to insulin that is
present at high levels during fetal development. It has been reported
that the receptor functions in signal transduction upon IGF-II binding
(for review, see Nishimoto et al.(1991)). In contrast, other
studies indicate that the IGF-II/MPR functions in clearance rather than
signal transduction and that IGF-II exerts its effects through the
IGF-I receptor and another uncharacterized receptor (Filson et
al., 1993; Liu et al., 1993; Baker et al.,
1993). Regardless, the IGF-II/MPR clearly mediates endocytosis of
IGF-II, resulting in its delivery to the lysosome and subsequent
degradation (Oka et al., 1985; Kiess et al., 1987a).
Thus, the receptor targets at least two classes of protein to the
lysosome.
Materials
The following buffers were used:
physiological binding buffer (PBB), 100 mM NaCl, 25 mM NaHCO, 4 mM KCl, 1.2 mM CaCl
, 1.2 mM NaH
PO
,
0.6 mM MgCl
, 0.3 mM MgSO
,
0.12 mM citric acid, 17 µM CuSO
, 10
µM NaF, 6 µM AlCl
, 22 nM NaI, 5 nM CoCl
, 45 nM MnCl
, 18 µM ZnSO
, 5.7 mM NaOH, 5 mM
-glycerophosphate, 20 mM HEPES,
pH 7.4, at 4 °C; acid buffer, 150 mM ammonium acetate, 250
mM acetic acid, pH 4.5; immunoprecipitation (IP) buffer, 50
mM Tris, pH 7.4, 0.5% bovine serum albumin, 0.02%
NaN
; modification buffer, 6 M guanidine-HCl, 2
mM EDTA, 50 mM Tris, pH 8.1; Lys-C buffer, 1 mM EDTA, 5% acetonitrile, 25 mM Tris, pH 8.5; glycosidase
buffer, 10 mM CaCl
, 20 mM sodium
cacodylate, pH 6.5; PBS, 137 mM NaCl, 2.7 mM KCl,
10.1 mM Na
HPO
, 1.76 mM
KH
PO
, pH 7.2; PBST, PBS with 0.05% Tween 20;
PBSB, PBS with 0.25% bovine serum albumin. When indicated, a protease
inhibitor mixture (PIC) was included at a final concentration of 1
mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 1
mM pepstatin. Fetal bovine serum was obtained from Sigma,
Hyclone, and Inovar. Bovine amniotic fluid, allantoic fluid, and urine
were generously provided by Drs. Darryl Byerley and Calvin Ferrell
(U.S. Department of Agriculture (USDA), Clay City, NE). Yeast
phosphomannan was very kindly provided by Dr. M. E. Slodki (USDA,
Peoria, IL). Phosphomannan affinity resin was prepared by coupling
cyanogen bromide-activated Sepharose CL-6B (Pharmacia Biotech Inc.) to
phosphomannan core as described previously (Sahagian et al.,
1982). Recombinant human IGF-II was generously provided by Dr. Yitzhak
Stabinsky (PeproTech Inc., Rocky Hill, NJ). Nitrocellulose was from
Schleicher and Schuell. All other chemicals were reagent grade.
Protein Purification
Either low quality or expired
lots of tissue culture grade fetal bovine serum were used for
purification of sIGF-II/MPR (see ). Typically, we used lots
that contained 4 µg of receptor/ml, but similar elution
profiles were obtained using lots that ranged from 2 to 6 µg of
receptor/ml. Serum (5 liters) was diluted with an equal volume of
PBB/PIC, filtered through 0.2-µm cellulose acetate membranes
(Millipore), and loaded onto a phosphomannan-Sepharose CL-6B affinity
column (5
10 cm) at 500 ml/h. After loading, the column was
disassembled, the resin batch was washed with 2 column volumes of PBB
(5 times), the column was reassembled, and the column was washed at 500
ml/h until A
reached base line. In early
preparations, the column was mock eluted at 30 ml/h with 2.5 mM glucose 6-phosphate in PBB/PIC to release nonspecifically bound
substances; this step was subsequently omitted as negligible amounts of
protein were released. The column was then eluted at 30 ml/h with 2.5
mM Man-6-P in PBB/PIC. Recovered proteins were concentrated by
ultrafiltration using a YM-100 membrane (Amicon Inc.). Gel filtration
chromatography at neutral pH was performed on affinity-purified protein
using two columns connected in series (Superose 12, Pharmacia; 1.6
85 cm; Superdex 75, Pharmacia; 1.6
60 cm) equilibrated
with 0.1 mM Man-6-P/PBB and run at 1.0 ml/min. Selected
fractions (e.g. eluting between 96-150 ml; Fig. 1)
from the neutral gel filtration chromatography step were concentrated
as above, acidified to pH 4.5 by the addition of 0.1 volumes of 10
acid buffer, and incubated overnight. Acidified protein was
rechromatographed at 0.25 ml/min on the gel filtration columns
equilibrated with 1
acid buffer. All steps were performed at 4
°C.
Figure 1:
Separation of
sIGF-II/MPR and bound ligands. A, gel filtration
chromatography was carried out as described under ``Experimental
Procedures.'' Upper part, pH 7.4, neutral pH
chromatography on the Man-6-P eluate from the phosphomannan column; lower part, pH 4.5, acidic pH chromatography on pooled
fractions (96-150 ml) from the neutral column. Absorbance at 280
nm was continuously monitored and recorded at 1 (dashedline) and 50
(solidline) scale
expansion. The minor A
absorbing peak eluting
after 240 ml in both chromatograms is likely to be salts since it was
of variable intensity in different runs and did not contain any
detectable protein. Positions of molecular mass standards run on the
neutral gel filtration columns are marked with arrowsabovetoppart (from left to right: thyroglobulin, 669 kDa; apoferritin, 443 kDa; myosin,
200 kDa; alcohol dehydrogenase, 150 kDa; bovine serum albumin, 67 kDa;
carbonic anhydrase, 29 kDa; ribonuclease A, 13.7 kDa; and aprotinin,
6.5 kDa). B, SDS-PAGE analysis of gel filtration
chromatography fractions. Samples (18 µl) of 6-ml column fractions
were resolved on a 5-20% polyacrylamide gel under reducing
conditions. Volume refers to the elution position of each fraction. The
gel was stained using Coomassie Blue, photographed, and then stained
using ammoniacal silver. Positions of molecular mass standards are
marked.
Precipitation of IGF-II
Samples were taken from a
single fraction of the A2 and A3 peaks (equivalent to the 200- and
235-ml elution volumes, respectively, of the acidic gel filtration
chromatogram in Fig. 1A) and radiolabeled using
NaI (DuPont NEN NEZ-033A) and IODO-GEN (Pierce) as
described previously (Valenzano et al., 1993). Approximately
150,000 cpm each of labeled peak A2 and A3 proteins were incubated with
either 40 ng of anti-IGF-II monoclonal antibody (Amano) or 10 µl of
sIGF-II/MPR-Sepharose (0.9 mg of sIGF-II/MPR per ml of resin) in a
final volume of 400 µl of IP buffer. Samples precipitated with
immobilized receptor had 10 mM Man-6-P included in the IP
buffer. All samples were mixed overnight at 4 °C in the absence or
presence of 60 ng of recombinant human IGF-II (rhIGF-II). Protein
A-Sepharose (Pharmacia) was added to the samples containing anti-IGF-II
and mixed at room temperature for an additional 30 min. After washing 5
times with IP buffer, pellets were resuspended in 80 µl of sample
buffer and analyzed by SDS-PAGE.
Reduction and Alkylation
Samples in 1 acid
buffer were dried and resuspended to
0.1-0.3 µg/µl
protein in modification buffer. Samples were reduced with 20 mM DTT and alkylated in the dark with either 40 mM
iodoacetic acid or iodoacetamide. Incubations were performed for 3 h at
50 °C under a N
atmosphere with continuous stirring.
For radiolabeling, samples were initially reduced with 2 mM DTT and alkylated with 4 mM iodo[2-
C]acetic acid (50 µCi; Amersham
Corp.) as above followed by 20 mM DTT and 40 mM iodoacetic acid to ensure complete alkylation. Reactions were
quenched with 150 mM
-mercaptoethanol for 15 min at room
temperature. Reaction mixtures were diluted with an equal volume of
water and applied to 0.1% trifluoroacetic acid-equilibrated Sep-Pak C18
cartridges (Waters). After washing with 10 ml of 0.1% trifluoroacetic
acid, protein was eluted with 70% acetonitrile, 20% 1-propanol, 0.1%
trifluoroacetic acid. Specific activities for radiolabeled high M
and 7.5-kDa IGF-II were
250 Ci/mol assuming
complete recovery of protein.
Analysis of IGF-II Binding Proteins in Fetal Bovine
Serum
Mature bovine IGF-II from peak A3 was iodinated using
lactoperoxidase as described previously (Daughaday, 1987). Radiolabeled
protein was purified on a Sephadex G50 fine column (Pharmacia, 0.7
28 cm) using PBSB as the mobile phase. Three radioactive peaks
were detected, and fractions comprising the second peak (IGF-II
monomer) were pooled. The specific activity was
10 Ci/mmol
assuming 50% recovery of IGF-II in the monomer peak. Radiolabeled
IGF-II (10 ng) was incubated with fetal bovine serum (1 ml) for 24 h at
room temperature. The serum was applied to a Superose 12 column (1.6
85 cm) and eluted at 1 ml/min with PBST at 4 °C. Fractions
(2 ml) were counted for 1 min in a Packard Cobra auto-
-counter.
Other Procedures
sIGF-II/MPR concentrations in
both purified and crude samples were determined by a two-antibody
sandwich enzyme-linked immunosorbent assay as described previously
(Chen et al., 1993). Protein concentrations were determined
with bovine serum albumin standards using the dye-binding assay
(Bradford, 1976) adapted to microtiter plates. SDS-PAGE was performed
as described (Laemmli, 1970). Gels were stained with 0.2% Coomassie
Brilliant Blue R-250 (Bio-Rad) and/or silver (Wray et al.,
1981; Merril et al., 1981) as indicated. Dried radioactive
gels were either imaged on Kodak X-Omat RP film or exposed to a
phosphor storage screen, scanned, and quantitated using a Molecular
Dynamics PhosphorImager 400 and ImageQuant 3.15 software. Sequencing
was performed using automated Edman degradation on an Applied
Biosystems Inc. model 475A gas phase sequencer with a 120A on-line
phenylthiohydantoin-derivative analyzer and a 900A data control
analysis module. The chromatograms were recorded and the results
analyzed using an Applied Biosystems 475A report generator.
Purification of sIGF-II/MPR and Bound
Ligands
This study was initiated to identify
non-Man-6-P-containing ligands that are associated with the receptor in vivo. A suitable source of sIGF-II/MPR was identified by
screening different bovine body fluids including serum, allantoic
fluid, amniotic fluid, and urine. The results are summarized in . The richest source was fetal serum, with receptor levels
in 23 different lots ranging from 1.8 to 7.9 µg/ml. Interestingly,
serum classified as unacceptable for tissue culture by different
suppliers (``low quality'') had lower receptor concentrations
than tissue culture grade serum ().
Characterization of Ligands
Protein blotting
experiments using an I-labeled sIGF-II/MPR probe
demonstrated that the 6- and 12-20-kDa bands are specifically
recognized by the receptor (Fig. 2). Furthermore, addition of
Man-6-P does not abolish binding, while IGF-II does. In contrast to
their Coomassie and silver staining intensities, there is a greater
signal seen for the 12-20-kDa bands than for the 6-kDa band. This
is misleading, since control experiments using iodinated tracers
demonstrate that significant amounts of all species are washed off the
membrane during the assay, with the 6-kDa band being lost
preferentially. In addition, the 6-kDa band has precisely the same
mobility as the 7.5-kDa rhIGF-II standard (Fig. 2). In a
complementary experiment using radiolabeled protein, the bands present
in peaks A2 and A3 were all precipitated by immobilized sIGF-II/MPR,
and this was inhibited by rhIGF-II (Fig. 3). In addition, all of
these radiolabeled bands were specifically recognized by an anti-IGF-II
monoclonal antibody (Fig. 3). Finally, sequence analysis showed
that >90% and >99% of the material present in representative
fractions of peaks A2 and A3, respectively, had the amino terminus
AYRPS. This is identical to that reported for mature bovine IGF-II
(Honegger and Humbel, 1986). Taken together, these data indicate that
the 12-20-kDa bands represent high molecular weight forms of
IGF-II and that the 6-kDa band represents mature 7.5-kDa bovine IGF-II.
Figure 2:
Activity blotting of gel filtration column
fractions. Peak fractions from the neutral and acidic gel filtration
columns were sampled (peaksN1 and A1, 0.1%
of total; peaksA2, A2`, and A3,
0.3% of total), and fractionated on 5-20% polyacrylamide gels
under nonreducing conditions. Protein was transferred to 0.2-µm
nitrocellulose and probed with 5 nMI-labeled
sIGF-II/MPR (Valenzano et al., 1993) in the presence or
absence of 2.5 mM Man-6-P or 1 µM human IGF-II as
indicated. The PhosphorImager printouts shown represent a 16-h exposure
and a color range of 1-1500 machine counts. Only the relevant
portions of the membranes are shown, since no signal was detected in
the higher molecular weight regions. Brackets and arrows represent the migration of high M
and 7.5-kDa
IGF-II, respectively.
Figure 3:
Precipitation of high M and 7.5-kDa IGF-II. Iodinated samples of peaks A2 and A3 were
incubated with either anti-IGF-II monoclonal antibody/protein
A-Sepharose or sIGF-II/MPR-coupled Sepharose CL-6B in the absence or
presence of rhIGF-II as described (see ``Experimental
Procedures''). Pellets were resuspended in reducing SDS-PAGE
sample buffer and resolved on a 5-20% polyacrylamide gel.
Proteins were visualized by autoradiography after a 24-h exposure. The
positions of high M
and 7.5-kDa IGF-II are
indicated.
In the course of characterizing these IGF-II species, we noticed
that their migration during SDS-PAGE was dramatically influenced by
different reduction and alkylation treatments. We systematically
investigated this using rhIGF-II (Fig. 4A). Both
nonreduced and DTT-treated rhIGF-II migrated with the 6-kDa standard (lanes1 and 4, respectively), while
carboxyamidation with iodoacetamide slightly retarded migration (lane3). In contrast, carboxymethylation with
iodoacetic acid greatly decreased migration, with the modified protein
now running just behind the 14-kDa standard (lane2).
The major band in peak A3 also exhibited this shift, precisely
comigrating with rhIGF-II under all conditions (Fig. 4B, lanes1, 2, 4, and 5, and
data not shown). These observations further confirm that peak A3
contains 7.5-kDa bovine IGF-II. The bands in peak A2 also show this
characteristic mobility shift upon iodoacetic acid modification (Fig. 4B, lanes3 and 6, and
data not shown). This supports our conclusion that these bands
represent different IGF-II species. The markedly decreased migration of
the carboxymethylated proteins may be a consequence of reduced SDS
binding or altered conformation due to introduction of the negatively
charged functional groups. Similar behavior has been reported
previously for other chemically modified or phosphorylated proteins
(Banker and Cotman, 1972; McCumber et al., 1976; Deibler et al., 1975). In addition, iodoacetic acid treatment is
useful in increasing resolution of the bands by SDS-PAGE: analysis on
12.5-17.5% acrylamide gels revealed that there were at least 12
distinct high M IGF-II proteins (Fig. 4C). This complexity was also evident using ion
exchange chromatography on the native proteins.
(
)
Figure 4:
Electrophoretic mobilities of high M and 7.5-kDa IGF-II. A, rhIGF-II was
modified as described (see ``Experimental Procedures''). Lane1, DTT-reduced; lane2, DTT
+ iodoacetic acid; lane3, DTT +
iodoacetamide; lane4, nonreduced. B,
rhIGF-II (lanes1 and 4), bovine 7.5-kDa
IGF-II (lanes2 and 5), and bovine high M
IGF-II (lanes3 and 6) modified with iodoacetic acid (lanes1-3) or iodoacetamide (lanes4-6). C, high M
IGF-II modified with iodoacetic acid. PanelsA and B represent Coomassie-stained 16% Novex gels. PanelC represents a 12.5-17.5% polyacrylamide
gel stained with silver. The high M
IGF-II in panelB was prepared by rechromatography of peak A2
on the Superdex 75 column to remove 7.5-kDa IGF-II. Chromatography
conditions were identical to the acidic gel filtration step described
in Fig. 1. Two fractions from the purified high M
IGF-II peak were pooled and used for modification. The high M
IGF-II in panelC was prepared
by sampling all peak A2 fractions to obtain an accurate representation
of the high M
IGF-II proteins and mature 7.5-kDa
IGF-II. The molecular mass standards for each panel are
shown.
To investigate factors that contribute to the
increased molecular weight and size heterogeneity of the high M IGF-II proteins, we adapted the strategy of
protease and glycosidase treatments used by Hudgins and colleagues
(Hudgins et al., 1992). Pooled fractions of high M
IGF-II and rhIGF-II standard were radiolabeled
at Cys-9, -21, -46, -47, -51, and -60 by reduction and alkylation with
iodo[2-
C]acetic acid. The proteins were digested
with Lys-C which is predicted to cleave pro-IGF-II after Lys-65, -88,
-96, -120, -129, and -151 (see Fig. 5). The rhIGF-II standard,
which terminates at Glu-67, showed a slight shift in mobility by
SDS-PAGE, consistent with the loss of its two COOH-terminal amino acids (Fig. 6). Incubation with the protease converted all the high M
IGF-II species into a single band that had the
same mobility as digested rhIGF-II. This demonstrates that the
increased molecular weight and size heterogeneity seen among the high M
IGF-II proteins is due to variations beyond
Lys-65.
Figure 5:
Schematic of prepro-IGF-II. Bovine IGF-II
is synthesized as a 179-amino acid preproprotein. After removal of the
signal sequence (hatchedregion), further processing
within the E peptide (stippledregion) occurs,
ultimately yielding the mature 67-residue protein. The enlarged region
shows potential sites for O-linked glycosylation (asterisks). The positions of the six lysine residues (65, 88,
96, 120, 129, and 151) are marked with arrows (see Fig.
6).
Figure 6:
Lys-C
digestion of high M and 7.5-kDa IGF-II. Duplicate
samples (1 µg) of reduced and
C-alkylated high M
and 7.5-kDa rhIGF-II were dried and resuspended
in 26 µl of Lys-C buffer. Endoproteinase Lys-C (0.4 µg;
Boehringer Mannheim) was added to each sample and incubated at 37
°C for 20 h. Digested samples were resolved on a 12.5-17.5%
polyacrylamide gel and visualized by autoradiography after a 2-week
exposure. Positions of molecular mass standards are indicated. High M
IGF-II was separated from 7.5-kDa IGF-II as
described in Fig. 4.
Pro-IGF-II has no consensus N-linked glycosylation
sites; however, numerous potential sites for O-linked
glycosylation are present (Fig. 5). To determine if variability
among the high M IGF-II species arises from this
modification, the mixture was digested with neuraminidase and O-glycanase to remove terminal sialic acids and O-linked core disaccharides, respectively (Fig. 7).
Treatment with both enzymes led to a collapse of nearly all high M
IGF-II species into a single band that had the
same mobility as the smallest high M
IGF-II and
likely represents extended, nonglycosylated IGF-II (arrow).
These data, together with the Lys-C data above, indicate that most of
the high M
forms of IGF-II contain an identical
COOH-terminal extension with variable O-linked glycosylation.
In addition, the high M
forms of IGF-II were also
digested with the two glycosidases separately. Treatment with
neuraminidase alone converts many of the upper bands into lower
molecular weight species, demonstrating that these proteins contain
sialic acid. In contrast, treatment with O-glycanase alone has
little effect on the higher molecular weight forms, while the band
running just above the deglycosylated form displays an increased
intensity. As O-glycanase only removes the unmodified
disaccharide Gal
1-3GalNAc from threonine or serine residues,
this suggests that at least some of the high M
IGF-II species contain both O-glycanase-sensitive and
resistant linkages, indicating glycosylation at two or more sites.
Figure 7:
Glycosidase treatment of high M IGF-II. Samples (0.8 µg) of reduced and
C-alkylated high M
IGF-II were dried
and incubated with either 10 milliunits of neuraminidase (Arthrobacter;
Boehringer Mannheim), 2 milliunits of O-glycanase (Genzyme),
or both enzymes in a total volume of 30 µl of glycosidase buffer.
Digests were carried out at 37 °C for 20 h. For double digests,
samples were first incubated with neuraminidase for 2 h at 37 °C
before addition of O-glycanase. Digested samples were resolved
on a 12.5-17.5% polyacrylamide gel and visualized by
autoradiography after a 2-week exposure. Positions of molecular mass
standards are indicated. High M
IGF-II was
separated from 7.5-kDa IGF-II as described in Fig.
4.
sIGF-II/MPR as an IGF-II Binding Protein
A large
proportion of the affinity-purified receptor contains bound IGF-II. In
a typical preparation from 5 liters of serum, we recover 18, 0.19, and
0.27 mg of protein in peaks A1, A2, and A3, respectively. (These values
were obtained using the dye-binding assay, but similar results were
found using quantitative amino acid analysis). Based on our recoveries
from peaks A1 and A3, at least 50% of the receptor contained bound
IGF-II, assuming that the protein molecular weights of sIGF-II/MPR and
mature IGF-II are 250,000 and 7,500, respectively, and that the
binding stoichiometry of IGF-II and receptor is 1:1 (Tong et
al., 1988). This is a conservative estimate, since it ignores the
contribution of mature IGF-II and high M
IGF-II
present in peak A2.
5% eluting as the uncomplexed form (Fig. 8, upperpanel, peak4;
also see below). However, tracer redistributes among the different
peaks as a function of time, shifting from low to high M
species (Fig. 8, upperpanel, compare peak3 with peak1). While biphasic
binding is also seen at 4 °C, the slow redistribution process has
not reached equilibrium even after 48 h (Fig. 8, middlepanel and inset).
Figure 8:
Effects of
time and temperature on the association of I-labeled
IGF-II with serum proteins. Upper and middlepanels, serum was incubated with radiolabeled IGF-II
tracer as indicated and fractionated by gel filtration chromatography
(see ``Experimental Procedures''). Insets,
radioactivity eluting in peak1 is expressed as
percentage of the total recovered radioactivity. Lower panel,
serum was preincubated as indicated and then incubated with
radiolabeled IGF-II tracer for 24 h at 4 °C. Positions of molecular
mass standards are marked with arrows above the toppanel (from left to right: sIGF-II/MPR,
250 kDa; alcohol dehydrogenase, 150 kDa; aprotinin, 6.5
kDa).
Additional control
experiments indicate that the redistribution process is not caused by
preferential degradation of the low M binding
proteins over time. Serum was preincubated at either 4 °C for 4 h
or 22 °C for 24 h, chilled to 4 °C, and then incubated with
tracer for 24 h. Both conditions yielded nearly identical binding
profiles (Fig. 8, lowerpanel). This strongly
suggests that no appreciable degradation occurs during the 24-h
incubation at 22 °C.
binding proteins that have relatively low affinity for IGF-II,
followed by a slow equilibration to higher affinity high M
binding proteins. Alternatively, the initial
binding could reflect the forward rate constants of the different
binding proteins, while the final distribution would reflect both the
forward and reverse rate constants. Regardless of the mechanism, the
process appears to plateau after
24 h at 22 °C (Fig. 8, upperpanel, inset), and the equilibrium
value should reflect the relative concentration and affinities of the
different binding proteins and hence the underlying distribution of
endogenous IGF-II (Frost and Pearson, 1953).
of the total
IGF-II.
Figure 9:
Contribution of sIGF-II/MPR as an IGF-II
binding protein. A, untreated serum. B, serum
depleted of sIGF-II/MPR by phosphomannan-agarose chromatography. C, nonspecific binding. Untreated FBS was co-incubated with
tracer IGF-II and a 1000-fold excess (1.3 µM) of cold
rhIGF-II. (All incubations with tracer were for 24 h at 22
°C.)
-galactosidase and IGF-II do,
despite having different microscopic binding sites (Kiess et
al., 1989). Similarly, potential new ligands bound to the serum
receptor could be displaced by the phosphomannan agarose and/or
endogenous IGF-II. In addition, potential new ligands may be present in
tissues other than serum. To address these possibilities, we screened
different tissue extracts for non-Man-6-P-containing ligands.
(
)Two approaches were used. First, extracts were
fractionated by SDS-PAGE, transferred to nitrocellulose, and probed
with the radiolabeled soluble receptor as described in Fig. 2.
Second, extracts were applied to immobilized sIGF-II/MPR columns in the
presence of Man-6-P to enrich for non-Man-6-P containing-proteins.
Bound material was eluted with an acidic buffer, fractionated by
SDS-PAGE, and screened as above. Both approaches yielded negative
results. Thus, although we cannot exclude the possibility that other
ligands exist, despite extensive effort, we have not detected them.
25% of the total IGF-II in fetal bovine serum is bound to soluble
receptor.
IGF-IIs do
not contain these internal substitutions. Recently, two IGF-II species
with apparent molecular weights of 15,000 and 11,500 were purified from
human serum Cohn fraction IV
and represent partially
processed forms of the 156-residue pro-IGF-II (Hudgins et al.,
1992). In addition, these 87-88-amino acid proteins are
glycosylated at Thr-75. It is likely that our glycosylated high M
IGF-II species represent additional isoforms of
these proteins.
IGF-II ligands,
allowing them to exert their effects. Second, the biological function
of the high M
IGF-II proteins and the purpose of
their glycosylation remain intriguing and unanswered questions. It has
been suggested that glycosylation is required for proper proteolytic
processing of pro-IGF-II to the mature 7.5-kDa form (Daughaday et
al., 1993). If so, the mechanism may involve binding of
glycosylated IGF-II to intracellular lectins that retain and present
the glycoprotein to the appropriate processing enzymes (Fielder and
Simons, 1994). In this case, the sIGF-II/MPR-bound high M
IGF-IIs may simply reflect inefficiencies in the
processing system. Alternatively, the glycosylated IGF-II species may
have unique biological functions. This is not unprecedented, since N-linked glycosylation of several glycoprotein hormones is
necessary for receptor activation but not binding (for review, see
Sairam(1989)). Similarly, the different glycosylated high M
IGF-II species may be targeted to specific
destinations or activate specific receptors. Future studies using
individually-purified high M
IGF-II glycoproteins
will be instrumental in determining their pharmacological and
biochemical properties.
Table: sIGF-II/MPR content of bovine body fluids
Table: sIGF-II/MPR as an IGF-II binding protein
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.