From the
The mannose 6-phosphate/insulin-like growth factor II receptor
(M6P/IGF-II receptor) binds insulin-like growth factor II (IGF-II) with
high affinity. To localize the IGF-II binding site within the 15
repeating units that form the extracytoplasmic domain of the receptor,
purified human M6P/IGF-II receptor was digested with thermolysin, and
the fragments were analyzed for their ability to bind
The mannose 6-phosphate/insulin-like growth factor II receptor
(M6P/IGF-II receptor)
The M6P/IGF-II receptor is a type I
transmembrane protein(6) . The extracytoplasmic domain is
composed of 15 repeating units, each of 134-167 amino acids in
length, which are homologous to each other and to the extracytoplasmic
domain of the 46-kDa mannose 6-phosphate receptor (MPR46)(7) .
Within the extracytoplasmic domain the M6P/IGF-II receptor binds 2 mol
of mannose 6-phosphate (8) and 1 mol of IGF-II (9)/mol of
receptor. The binding of IGF-II may be a characteristic of mammals,
that has been demonstrated so far for the receptors from human
(10-13), bovine(9, 14) , opossum(15) , and
rat(14, 16) , while the receptors from chicken (17, 18) and frog (18) lack the binding of
IGF-II.
IGF-II is a single-chain polypeptide of 67 amino acids, that
shares a high degree of homology with IGF-I and insulin(19) .
The majority of extracellular IGF-II is bound to IGF-binding proteins
and acts as a mitogen for different cell types (20) and as a
fetal growth factor (21). IGF-II binds to the M6P/IGF-II receptor with
a K
In the present study, we characterized two
IGF-II-binding fragments derived from human M6P/IGF-II receptor
digested with thermolysin. The sequence analysis of the fragments
revealed that each fragment consists of several peptides, cross-linked
by disulfide bridges. The peptides of the smaller of the two fragments
comprise the C terminus of repeat 9, the entire repeat 10, and the N
terminus of repeat 11. In a complementary approach truncated forms of
the human M6P/IGF-II receptor fused to the extracytoplasmic domain of
the MPR46 were expressed and tested for their ability to bind IGF-II.
Whereas the fusion proteins encoding repeat 10 alone did not bind
IGF-II, fusion proteins containing repeats 10-15, 11-15,
and 10-11 bound IGF-II. Taking the results of both approaches
together, we conclude that the N terminus of repeat 11 comprising amino
acids 1508-1566 contains the binding site for IGF-II.
Fused
to the 3` site of the MPR46 cDNA, the constructs contain the indicated
truncated human M6P/IGF-II receptor cDNAs(6) . Construct
10-15 contains nucleotides 4246-7028 with a stop codon in
position 7027, coding for amino acids 1367-2293 (repeat
10-15); construct 10 contains nucleotides 4246-7028 with a
stop codon in position 4693, coding for amino acids 1367-1515
(repeat 10); construct 10-11 contains nucleotides 4246-7028
with a stop codon in position 5098, coding for amino acids
1367-1650 (repeat 10-11); and construct 11-15
contains nucleotides 4669-7028 with a stop codon in position
7027, coding for amino acids 1508-2293 (repeat 11-15).
These truncated M6P/IGF-II receptor cDNAs were generated using a KpnI/KpnI fragment comprising nucleotides
3242-7851 of the M6P/IGF-II receptor cDNA in M13mp18 plasmid as
template for site-directed mutagenesis according to the protocol of
Nakamaye and Eckstein(34) . BglII sites were created at
the 5` end of repeat 10 or 11 and at the 3` end of the stop codons
listed above. The BglII/BglII fragments were cloned
into the MPR46/pBEH plasmid opened by BamHI. In each construct
the fusion was represented by amino acid 190 of MPR46 and the first
amino acid of either repeat 10 (threonine 1367) or repeat 11
(methionine 1508) of M6P/IGF-II receptor. For each construct the
presence of the mutations was confirmed by fluorescent dye terminator
cycle sequencing and analysis on an automated DNA sequencer (Applied
Biosystems). Oligonucleotide primer for mutagenesis and sequencing were
synthesized using an 381A solid-phase synthesizer (Applied Biosystems)
and purified on a MonoQ anion exchange column (Pharmacia).
Peptides 23B, 23C, 37B, and 37D were sequenced up to their
C termini. The C termini of peptide 23A and 37A, which share common
N-terminal sequences, could not be identified. However, they extend
beyond the cycle 19 given in Fig. 4, as indicated by signals in
cycle 22 (leucine) and 23 (serine).
Peptide 23B terminates in front
of a thermolysin cleavage site (N-terminally of valine 1341). For
peptide 37B we assume that it consists of 11 rather than 10 residues
(as indicated by the sequence analysis) and that the C-terminal residue
(proline 1340) was washed out, as often observed in automatic Edman
degradation. With this assumption peptide 37B is identical to peptide
23B. Additionally, peptide 37D and, with the assumption of a washout of
the C-terminal glycine 1562, peptide 23C terminate in front of a
thermolysin cleavage site (leucine 1698 and valine 1563).
For the
alignment of the peptides, it should be noted that nonreducing
conditions were used in all experimental steps. Receptor peptides
comigrating in SDS-PAGE are therefore assumed to be linked by disulfide
bonds. According to the model of Lobel et al.(7) , for
disulfide bonding of the receptor, peptide 23B is expected to be
connected with peptide 23A by a disulfide bridge between cysteines 1333
and 1361 (see Fig. 5). To allow disulfide binding of peptide 23A
with a following peptide of the 23-kDa fragment, we have to postulate
that peptide 23A extends at least up to cysteine 1516, to allow its
disulfide binding to cysteine 1553 of peptide 23C. Peptide 23C contains
a second cysteine residue (cysteine 1559), which is proposed to be
linked in the receptor to cysteine 1566. The sequence data are
compatible with a linkage of cysteine 1566 to the dipeptide alanine
1565-cysteine 1566, which can be cut out by thermolysin, while more
extended sequences for cysteine 1566 containing peptides are excluded
by the sequence analysis. The molecular mass of the 23-kDa
IGF-II-binding fragment calculated from the sequenced peptides is
20.3-23.9 kDa, if the C terminus of peptide 23A is assumed to be
at one of the thermolysin cleavage sites between cysteine 1516 and
peptide 23C. This value fits well to the determined molecular mass of
23 kDa. Altogether the 23-kDa fragment consists of three peptides (plus
the unidentified dipeptide alanine-cysteine, see above) connected to
each other by disulfide bridges. The peptides extend from amino acid
1331 to 1562 (or 1566), which correspond to the C terminus of repeat 9,
the entire repeat 10, and about 40% of the N terminus of repeat 11.
The peptides recovered from the 37-kDa fragment can be interpreted
in an analogous way (see Fig. 5). Peptide 37B, which is identical
to peptide 23B, and peptide 37A are connected by the disulfide bridge
between cysteine 1333 and 1361. Peptide 37A is predicted to be
disulfide bounded to peptide 37D via cysteine 1652 and cysteine 1695.
The calculated molecular mass of the 37-kDa fragment is 34.9 kDa, if
the C terminus of peptide 37A is assumed to be at the first thermolysin
cleavage site following cysteine 1652 (N-terminally of valine 1654).
The calculated value of 34.9 kDa and the determined molecular mass of
37 kDa of the purified fragment agree well. Thus, the 37-kDa fragment
consists of three disulfide-connected peptides extending from amino
acid 1331 to 1697, which corresponds to the C terminus of repeat 9, the
entire repeats 10 and 11, and the N-terminal third of repeat 12.
We selected two different strategies to localize the binding
site for IGF-II at the M6P/IGF-II receptor. In our first approach we
purified two proteolytic receptor fragments of 23 and 37 kDa, which
retained their ability to bind IGF-II. The amino acid sequences of this
fragments overlap in the region of the C terminus of receptor repeat 9,
the entire repeat 10, and the N-terminal 40% of repeat 11, indicating
that this sequence of M6P/IGF-II receptor (amino acids 1331-1566)
is sufficient for IGF-II binding.
Garmroudi and MacDonald (40) recently published a study on the localization of the
IGF-II cross-linking site of the rat and bovine M6P/IGF-II receptor. In
contrast to the approach used in this study, binding and cross-linking
to IGF-II was carried out before the receptor was proteolytically
fragmented. Several
In our second approach we expressed truncated forms
of the M6P/IGF-II receptor in cells and analyzed their ability to bind
IGF-II. We could show that three fusion proteins containing repeats
10-15, 10-11, and 11-15 could be cross-linked to
IGF-II, demonstrating that repeat 11 (amino acids 1508-1650) is
sufficient for binding of IGF-II. The value of 10 nM unlabeled
IGF-II leading to half-maximal inhibition of
In a related study Dahms et al.(24) analyzed
truncated forms of the bovine M6P/IGF-II receptor transiently expressed
in COS cells. They found that a construct encoding repeats 5-11
could be cross-linked to IGF-II, whereas a construct encoding repeats
5-10 failed to be cross-linked. These results showed that the
presence of repeat 11 is necessary for the cross-linking to IGF-II. It
remained unknown, however, whether repeat 11 is sufficient for IGF-II
binding or whether one or several of repeats 5-10 are required to
form the IGF-II binding site.
Taking the results of our two
approaches, we can conclude that the N-terminal 40% of repeat 11,
comprising amino acids 1508-1566, contains the binding site for
IGF-II. Since IGF-II also binds to the IGF-I receptor, the insulin
receptor(41) , and IGF-binding proteins (42), we searched for
sequence homology between the IGF-II binding site of the M6P/IGF-II
receptor residues 1508-1566 and the other IGF-II-binding
polypeptides. We did not find an obvious homology with the IGF-I and
the insulin receptor. This is not unexpected, as the amino acid
residues of IGF-II mediating the binding to the M6P/IGF-II receptor and
the other two receptors are located on different sites of the IGF-II
molecule(43, 44) . The amino acid residues critical for
binding of IGF-II to IGF-binding proteins form a patch that partly
overlaps with the patch formed by the amino acid residues essential for
binding to the M6P/IGF-II receptor(43, 44) . In spite of
the overlapping binding sites at the ligand, obvious sequence
homologies between the IGF-II binding sites at the M6P/IGF-II receptor
and the IGF-binding proteins were not detectable.
In summary our
data suggest that the N-terminal 40% of M6P/IGF-II receptor repeat 11
is sufficient to form the binding site for IGF-II. All M6P/IGF-II
receptor constructs were expressed N-terminally fused to the
extracytoplasmic domain of the MPR46. It should therefore be tested
whether repeat 11 or a fragment of it would form a IGF-II binding site
when expressed as such.
Cross-linking of
M6P/IGF-II receptor and fusion proteins to
We thank Dr. W. S. Sly for providing the cDNA of the
human M6P/IGF-II receptor, Kirsten Theiling and Wolfgang Lehnert for
their help in preparing the DNA constructs, and Dr. R. Pohlmann for
preparing the MPR46/pBEH plasmid. We thank Klaus Neifer for assistance
during amino acid sequencing.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-IGF-II in a cross-linking assay. Two IGF-II-binding
receptor fragments of 23 and 37 kDa were purified. Sequence analysis
revealed that the fragments consist of disulfide connected peptides
comprising amino acids 1331-1566 and 1331-1697 of the
receptor repeats 9-12. In a second approach we expressed
truncated forms of the M6P/IGF-II receptor fused to the C terminus of
the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor.
Fusion proteins containing M6P/IGF-II receptor repeats 10-15,
10-11, or 11-15 bound IGF-II, whereas a fusion protein
containing the single repeat 10 failed to bind. This result indicates
that repeat 11 (amino acids 1508-1650) is sufficient for binding
of IGF-II. Residues 1508-1566, which are shared by the 23-kDa
IGF-II-binding fragment and repeat 11, are proposed to form the IGF-II
binding site of the M6P/IGF-II receptor.
(
)is a 300-kDa
transmembrane protein, which contains binding sites for two different
types of ligands in its extracytoplasmic domain. On the one hand,
lysosomal enzymes can bind via the mannose 6-phosphate residues of
their oligosaccharide side chains to the receptor. This binding is
critical for targeting of lysosomal enzymes from the Golgi or the
plasma membrane to the lysosomes(1, 2, 3) . On
the other hand, the receptor can bind the non-glycosylated insulin-like
growth factor II (IGF-II)(4) . Binding at the plasma membrane is
followed by internalization and degradation of IGF-II in the
lysosomes(5) .
of 0.2-14.5
nM(9, 14, 15) at a single binding
site, which is different from the two binding sites for mannose
6-phosphate(9, 11, 12, 14) , but the
influence of the binding of the one ligand on the affinity for the
other ligand remains still unclear. The exact locations of the binding
sites for mannose 6-phosphate and IGF-II still need to be defined.
Studies using truncated forms of the extracytoplasmic domain of the
bovine M6P/IGF-II receptor indicate that for binding of mannose
6-phosphate repeats 1-3 and 7-9(22, 23) ,
and for binding of IGF-II repeats 5-11 (24) are
sufficient.
Reagents
Recombinant human IGF-II was obtained
from Dr. W. Märki (Ciba-Geigy, Basel, Switzerland) and iodinated
with NaI to a specific activity of about 10 mCi/mg with
the aid of chloramine T as described(25) . Thermolysin, trypsin,
subtilisin, and V8 protease were from Sigma, and disuccinimidylsuberate
(DSS) from Pierce. Enzymes used for in vitro mutagenesis were
from New England Biolabs. Na
I and
[
S]methionine (>800 Ci/mmol) were purchased
from Amersham Corp. The anti-MPR46 antisera and the monoclonal
anti-MPR46 antibody 10C6 have been described
previously(26, 27) . All other reagents were of
analytical grade.
Purification and Digestion of the M6P/IGF-II
Receptor
The M6P/IGF-II receptor was purified from human
placenta as described previously(28) . The purified receptor was
dialyzed overnight against 50 mM imidazole, pH 7.0, 0.05%
Triton X-100 and precipitated with acetone (80% final concentration) at
-20 °C overnight. After centrifugation at 30.000 g for 45 min at 4 °C the pellet was dried under vacuum and
dissolved in 20 mM NaP
, pH 7.5, 20 mM NaCl to a final concentration of 0.17 mg/ml. In an analytical
scale, 3-µg aliquots of the receptor were incubated with 1, 2, 10,
and 20% (w/w) thermolysin, trypsin, subtilisin, or V8 protease in 24
µl of 20 mM NaP
, pH 7.5, 20 mM NaCl
at 37 °C for 16 h. The digestion was stopped by the addition of 10
mM EDTA, and the receptor fragments were analyzed in a IGF-II
cross-linking assay.
Purification of IGF-II-binding M6P/IGF-II Receptor
Fragments
For purification of IGF-II-binding fragments, three
aliquots of 2.0-2.3 mg M6P/IGF-II receptor were incubated with 2%
(w/w) thermolysin at 37 °C for 16 h as described above. After the
addition of EDTA, lyophilization, and dissolving in 600 µl of
water, each aliquot was loaded on a Superdex HR200-size exclusion
column (Pharmacia Biotech Inc.) equilibrated with 20 mM
NaP, pH 7.5, 20 mM NaCl. The receptor fragments
were eluted from the column with a flow rate of 0.5 ml/min and
collected in fractions of 2 ml. One µl of each fraction was
analyzed in the IGF-II cross-linking assay. Fractions 42-47 (pool
I) and fractions 49-54 (pool II) of the three runs were combined,
lyophilized, and dialyzed against 5 mM Tris/HCl, pH 7.5, 0.1%
SDS overnight. The dialyzed samples were loaded on a preparative
SDS-polyacrylamide gel (10% acrylamide for pool I and 12% for pool II).
Following electrophoresis a small strip of the gel was cut off and
stored. The fragments in the remaining gel were blotted on a
polyvinylidene difluoride (PVDF) membrane, stained with 0.2% Coomassie
Blue, 50% methanol, 10% acetic acid for 15 min, and washed three times
with 45% methanol, 7% acetic acid for 5 min. The stained receptor
fragments were subjected to an N-terminal sequence analysis. The gel
strip was cut into small strips, which were homogenized in 200 µl
of 5 mM Tris/HCl, pH 7.5, 0.1% SDS. The receptor fragments
were extracted by incubation at 37 °C overnight. After
centrifugation in an Eppendorf centrifuge, the supernatant was
lyophilized and the receptor fragments precipitated by an incubation in
500 µl of 85% acetone, 5% trimethylamine, 5% acetic acid, 5% water
for 1 h at 4 °C(29) . After centrifugation, the pellet was
dissolved in 30 µl of 20 mM NaP
, pH 7.5, 20
mM NaCl and analyzed in the IGF-II cross-linking assay.
Cross-linking of IGF-II to the M6P/IGF-II Receptor and
Its Fragments
Cross-linking of IGF-II to the M6P/IGF-II receptor
was carried out according to the procedure of Waheed et al. (12). One µg of the dialyzed and precipitated M6P/IGF-II
receptor or 3 µg of the receptor fragment mixture or aliquots of
purified fragments or dialyzed medium containing the fusion proteins
were incubated with 1-4 10
dpm
I-IGF-II in 30-60 µl of 20 mM NaP
, pH 7.5, 20 mM NaCl, 0.05% Triton X-100
overnight at 4 °C. To estimate the half-maximal inhibition of
cross-linking of
I-IGF-II to the fusion proteins, binding
and cross-linking were carried out in the presence of 10
to 10
M unlabeled IGF-II. After
binding cross-linking to
I-IGF-II was accomplished by
adding 0.06 mM DSS and incubation for 15 min on ice. The
reaction was stopped by the addition of 125 mM Tris/HCl, pH
6.8, 1% SDS, 10% glycerol and an incubation at 95 °C for 5 min. The
samples were subjected to SDS-PAGE under non-reducing conditions
according to the protocol of Laemmli et al.(30) .
Cross-linked
I-IGF-II was detected by autoradiography and
quantified by densitometry of the x-ray film.
Amino Acid Sequencing
In the area of the Coomassie
Blue-stained bands, the PVDF membrane was cut off, washed with 20%
methanol, and subjected to N-terminal sequencing as described
previously(31) . The determined amino acid sequences were
compared with the amino acid sequences of the M6P/IGF-II receptor and
thermolysin by means of the computer program GPMAW (Lighthouse Data,
Odense, Denmark).
Generation of MPR46-M6P/IGF-II Receptor
Constructs
Four constructs were generated coding for different
M6P/IGF-II receptor repeats fused to the extracytoplasmic domain of
human MPR46. All constructs contain the MPR46 cDNA coding for the
extracytoplasmic domain of MPR46 (nucleotides 1-715, amino acids
1-190, Ref. 32) inserted in the pBEH expression
vector(33) . To generate this MPR46/pBEH plasmid, a BamHI site was inserted into the MPR46 cDNA at position 710
and the EcoRI/BamHI fragment encoding nucleotides
1-715 was cut out and subcloned into the pBEH plasmid.
Tissue Culture and DNA Transfection
BHK cells
(clone 21) were transfected with 20 µg of expression vector as
described previously(35) . Stable colonies were transferred to a
24-well tissue culture plate. When cells were grown to confluence, the
rate of expression of the fusion proteins into the medium was
determined by an enzyme-linked immunoassay using anti-human MPR46
antiserum as described previously(26) . Cell colonies expressing
the fusion proteins were transferred to 35-mm cell culture dishes for
metabolic labeling and immunoprecipitation. For Western blot and IGF-II
binding analysis, cells were transferred to 75-cm cell
culture flasks. After cells had grown to confluence they were starved
for 24 h in 10 ml of serum-free medium. Medium was collected, dialyzed
against 20 mM NaP
, pH 7.5, 20 mM NaCl,
and concentrated to 200 µl by ultrafiltration. Equal amounts of
media were used for Western blot and IGF-II binding analysis.
Metabolic Labeling, Immunoprecipitation, and PMP Affinity
Chromatography
Confluent cells in 35-mm cell culture dishes were
starved for 1 h in methionine-free medium and labeled with 0.8 ml of
methionine-free medium containing 2.5% dialyzed fetal calf serum and 40
µCi of [S]methionine for 24 h. Medium was
collected and dialyzed against 50 mM Tris/HCl, pH 7.5, 150
mM NaCl at 4 °C and subjected to immunoprecipitation as
described previously (36) using anti-human MPR46
antiserum(26) . Binding of
S-labeled secretions
containing fusion proteins to PMP-Sepharose was carried out as
described previously(37) .
Western Blot Analysis
Aliquots of 5-100
µl of dialyzed and concentrated cell medium containing the fusion
proteins were subjected to Western blot analysis with ECL detection as
described previously (38) using the monoclonal anti-human MPR46
antibody 10C6 (27).
IGF-II Binding to the M6P/IGF-II Receptor and Its
Proteolytic Fragments
To examine the binding of IGF-II to the
M6P/IGF-II receptor purified from human placenta, a cross-linking assay
was performed. After incubation of the receptor with I-IGF-II, the mixture was subjected to cross-linking with
DSS, SDS-PAGE and autoradiography. A broad signal in the range of
250-300 kDa was detected (Fig. 1, lane2), which resolves upon shorter exposure into distinct
bands that coincide with bands observed when the gel was stained by
silver or a polyclonal receptor antibody (data not shown). When
I-IGF-II was subjected to cross-linking in the absence of
the receptor (Fig. 1, lane1), or when the
receptor was incubated with
I-IGF-II in the presence of
an excess of unlabeled IGF-II (lane3), the products
of 250-300 kDa disappeared, thus identifying the latter as
M6P/IGF-II receptor-
I-IGF-II cross-link products.
Figure 1:
Cross-linking of I-IGF-II
to M6P/IGF-II receptor and its fragments. M6P/IGF-II receptor (1
µg) purified from human placenta was cross-linked to
I-IGF-II via the cross-linker DSS and analyzed by
SDS-PAGE (10% acrylamide) under nonreducing conditions and
autoradiography as described under ``Materials and Methods'' (lane2). As controls cross-linking was carried out
in the presence of 10
M unlabeled IGF-II (lane3) or in the absence of receptor (lane1). Fragments derived from an incubation of 3 µg of
M6P/IGF-II receptor for 16 h with 2% thermolysin were cross-linked to
I-IGF-II in the absence (lane4) or
presence (lane5) of 10
M unlabeled IGF-II or in the absence of receptor (lane6). The apparent molecular masses of the cross-link
products are indicated on the left.
In
order to localize the IGF-II binding site within the receptor,
M6P/IGF-II receptor was incubated with different proteases, including
trypsin, thermolysin, subtilisin, and V8 protease. The proteolytic
fragments were analyzed for IGF-II binding. Thermolysin was found to
produce a series of products that retain IGF-II binding activity. Fig. 1(lane4) shows the pattern of
IGF-II-binding fragments, obtained after incubation of M6P/IGF-II
receptor for 16 h with 2% (w/w) thermolysin. A major 37-kDa and two
minor 33- and 23-kDa I-IGF-II cross-linked products were
detectable. When the digest was incubated in the presence of unlabeled
IGF-II, the binding of
I-IGF-II was inhibited completely (Fig. 1, lane5). Thermolysin or its
autocatalytic fragments did not bind
I-IGF-II (Fig. 1, lane6). The cross-linked products of
23-37 kDa are likely to contain receptor fragments of 1-2
repeats (the average mass of one repeat is about 16 kDa).
Purification of IGF-II-binding M6P/IGF-II Receptor
Fragments
In order to identify the IGF-II-binding fragments by
direct amino acid sequencing, 6.5 mg of M6P/IGF-II receptor were
digested with thermolysin and the fragments purified by a combination
of size exclusion chromatography and preparative SDS-PAGE. The elution
profile of the size exclusion chromatography (Fig. 2A)
indicates that the receptor was preferentially degraded to small
fragments with molecular masses of less than 15 kDa. The fractions were
analyzed for binding of IGF-II (Fig. 2B). The
IGF-II-binding fragments were detected in fractions 40-54, which
correspond to molecular masses of 30-50 kDa.
Figure 2:
Purification of IGF-II-binding
M6P/IGF-II receptor fragments by size exclusion chromatography. 6.5 mg
of M6P/IGF-II receptor were digested with 2% thermolysin in three
aliquots of 2.0, 2.2, and 2.3 mg and separated on a Superdex HR200-size
exclusion column in three runs as described. A, the elution
profile of the separation of 2.0 mg of receptor fragments is shown.
Elution (arrows) and molecular masses in kDa of standard
proteins are indicated. B, 1 µl of each fraction was
subjected to cross-linking to I-IGF-II, SDS-PAGE (10%
acrylamide), and autoradiography. The apparent molecular masses of the
cross-linked products (on the left) and the number of
fractions analyzed (below) are indicated. Fractions 42-47 (pool
I) and 49-54 (pool II) of the three runs were
combined.
Fractions
42-47 (yielding the majority of cross-linked products of 33 and
37 kDa) and fractions 49-54 (yielding the 23-kDa cross-linked
product) were pooled separately for further purification by SDS-PAGE.
In preliminary experiments, we showed that the receptor and its
fragments retain their ability to bind IGF-II after SDS-PAGE and
extraction from the gel, if SDS is removed by an acid acetone
precipitation. Following separation on a preparative SDS-polyacrylamide
gel, a small strip of the gel was cut off for cross-linking analysis
(see below). The remaining gel was blotted on a PVDF membrane, followed
by Coomassie Blue staining. Fractions 42-47 (Pool I) contained
four stainable fragments with the apparent masses of 37, 40, 45, and 63
kDa, and fractions 49-54 (Pool II) contained five stainable
fragments with apparent masses of 23, 25, 27, 33, and 37 kDa (see Fig. 3A). To test the ability of these fragments to bind
IGF-II, each gel strip was cut into eight small strips. After
extraction with 0.1% SDS and removal of SDS by an acid acetone
precipitation, binding of IGF-II was analyzed. In Pool I extracts
containing the 37- and 40-kDa Coomassie Blue-stainable fragments
yielded I-IGF-II cross-link products of 33-37 kDa
(data not shown). In Pool II the extract containing the 23-kDa
Coomassie Blue-stainable fragment yielded an
I-IGF-II
cross-link product of 23 kDa (Fig. 3B, lane4), and the extracts containing the 33- and 37-kDa
stainable fragments cross-linked products of 33 and 37 kDa (Fig. 3B, lanes1 and 2). A
weak signal observed in lane5 could not be assigned
to a Coomassie Blue-stainable fragment. Altogether, three
IGF-II-binding fragments with apparent masses of 23, 37, and 40 kDa
could be isolated from the thermolysin digest of the receptor.
Figure 3:
Cross-linking of I-IGF-II to
purified M6P/IGF-II receptor fragments. IGF-II-binding M6P/IGF-II
receptor fragments collected after size exclusion chromatography (see
Fig. 2, pool II) were purified by preparative SDS-PAGE (12%
acrylamide). Following electrophoresis one part of the gel was blotted
on a PVDF membrane and stained with Coomassie Blue. The other part of
the gel was cut into eight small strips, which were extracted with 0.1%
SDS. After removal of SDS by an acid acetone precipitation, the
extracted fragments were subjected to cross-linking to
I-IGF-II. PartA, the PVDF membrane
containing the Coomassie Blue-stained receptor fragments is shown. On
the left the apparent molecular masses of the fragments are
indicated in kDa. On the right the numbers and positions where
the strips were cut from the gel are indicated. PartB, the
I-IGF-II cross-linking analysis of
the extracts from the eight gel strips is shown. The number of gel
strips (above) and the apparent molecular masses of the
cross-linked products (on the left) are
indicated.
Sequencing of the IGF-II-binding Fragments
The
PVDF membranes containing the 23-, 37-, or 40-kDa IGF-II-binding
fragments were subjected to sequence analysis by automatic Edman
degradation. The results of the sequence analysis of the 23- and 37-kDa
fragment are shown in Fig. 4. Sequencing of the 40-kDa fragment
failed to provide any signal. Sequence analysis of the 23-kDa fragment
showed one to seven amino acid signals per cycle. The computer analysis
of this data revealed, that the amino acids could be assigned to the
N-terminal sequences of three M6P/IGF-II receptor peptides (sequence
23A: amino acids 1360-1378; 23B: 1331-1339; 23C:
1552-1561) (see Fig. 5) and the N terminus of thermolysin
(sequence 23E). The remaining signals (X) could neither be
assigned to M6P/IGF-II receptor nor to thermolysin.
Figure 4:
Sequencing of the IGF-II-binding
M6P/IGF-II receptor fragments. Sequencing of the IGF-II-binding
M6P/IGF-II receptor fragments of 23 and 37 kDa yielded several amino
acid signals per cycle. By computer analysis, the amino acids could be
assigned to the N-terminal sequences of human M6P/IGF-II receptor
peptides (23A-23C, 37A, 37B, and 37D) and to the N-terminal
sequence of thermolysin (23E). Some weak amino acid signals could not
be assigned (X). The sequencing yield (in pmol) is indicated
below each amino acid (, an exact quantification is not possible;
-, amino acid is not detectable). It should be noted that serine
and threonine cannot be quantified exactly, and cysteine cannot be
determined during sequencing by Edman
degradation.
Figure 5:
Alignment of the IGF-II-binding peptides
with the sequence of the M6P/IGF-II receptor. The amino acid sequence
of the human M6P/IGF-II receptor in the region of repeats 9-12 is
shown. Sequences determined by sequence analysis are labeled by grayboxes and labeled at the top. Disulfide bridges
following the model of Lobel et al. (7) are indicated. The
number of cysteines within the complete receptor sequence are partly
indicated.
Sequencing of
the 37-kDa fragment resulted in one to five amino acid signals per
cycle, which could be assigned to the N-terminal sequences of three
peptides derived from the M6P/IGF-II receptor (sequence 37A: amino acid
1360-1378; 37B: 1331-1340; 37D: 1692-1697) (see Fig. 5). The remaining signals (X) could not be
assigned.
Expression and IGF-II Binding of Truncated Forms of the
M6P/IGF-II Receptor
The IGF-II-binding M6P/IGF-II receptor
fragments of 23 and 37 kDa contain amino acid sequences, which are
located in repeats 9-12. To analyze, which of these receptor
repeats are involved in the binding of IGF-II, we expressed different
truncated forms of the M6P/IGF-II receptor in BHK cells (Fig. 6).
To increase the probability of a correct folding and disulfide
connection of the truncated receptor forms, all recombinants were
designed to terminate at the C-terminal amino acids of the receptor
repeats as defined by Lobel et al.(7) . The receptor
repeats were expressed in eucaryotic cells as fusion proteins connected
to the C terminus of the extracytoplasmic domain of the MPR46. This
strategy was chosen for the following reasons. 1) We speculated that
folding of a single M6P/IGF-II receptor repeat would possibly be
facilitated by expressing it as a fusion protein with a second receptor
repeat. As a repeat, which does not bind IGF-II and yet may assist
folding we chose the extracytoplasmic domain of the MPR46. The
extracytoplasmic domain of the MPR46 shows a sequence homology of
14-28% to each of the 15 repeats, which form the extracytoplasmic
domain of the M6P/IGF-II receptor(7) . This includes the
conservation of six cysteines proposed to form disulfide bonds within
each repeat. 2) The extracytoplasmic domain of the MPR46 provides a
N-terminal signal peptide, which guarantees the translocation of the
fusion proteins into the lumen of the endoplasmic reticulum. Since the
constructs lack the transmembrane domains of the MPR46 and the
M6P/IGF-II receptor, the fusion proteins should behave as secretory
proteins. 3) The extracytoplasmic domain of the MPR46 serves as a tag
for immunological detection of the fusion proteins and for binding to a
PMP affinity column due to the mannose 6-phosphate binding site in the
MPR46 domain(39) .
Figure 6:
MPR46-M6P/IGF-II receptor fusion proteins.
The four fusion proteins consist of the extracytoplasmic domain of the
human MPR46 (graybox) fused to the N terminus of
variable human M6P/IGF-II receptor repeats (whiteboxes). The numbers of the N- and C-terminal M6P/IGF-II
receptor amino acids are indicated.
The DNA constructs were cloned into the
expression vector pBEH and transfected into BHK cells. To isolate the
fusion proteins, cells were incubated for 24 h in serum-free medium.
After concentration and dialysis, the fusion proteins were analyzed by
Western blot with an antibody against MPR46 and by chromatography on a
PMP-Sepharose column in a mannose 6-phosphate-dependent manner (data
not shown). These results demonstrated that the MPR46 domain of the
fusion proteins folded correctly. Binding of IGF-II was tested in the
cross-linking assay with I-IGF-II.
M6P/IGF-II Receptor Repeats 10-15 Bind
IGF-II
Four fusion proteins containing different repeats of the
M6P/IGF-II receptor were expressed (Fig. 6). In our first
approach, we expressed repeats 10-15 (amino acids 1367-2293
of the M6P/IGF-II receptor) fused to the extracytoplasmic MPR46 domain.
Western analysis of secretions of cells expressing the fusion protein
10-15 revealed a 135-kDa polypeptide (Fig. 7A, lane2). Its mass fits well to the theoretical mass
of 138 kDa (105 kDa for repeats 10-15 and 33 kDa for the MPR46
extracytoplasmic domain). This calculation does not regard N-linked oligosaccharides possibly connected to one or more of
the six potential N-glycosylation sites of repeats
10-15. When binding of IGF-II to the fusion protein 10-15
was analyzed, a cross-link product of 145 kDa was detected (Fig. 7B, lane2). Its mass
corresponds to the calculated mass of the protein cross-linked to
IGF-II (138 + 7.5 kDa). Half-maximal inhibition of I-IGF-II cross-linking was observed at about 10 nM unlabeled IGF-II (). The results demonstrate that
repeats 10-15 are sufficient for binding of IGF-II.
Figure 7:
Western blot and I-IGF-II
cross-linking of the MPR46-M6P/IGF-II receptor fusion proteins. PartA, concentrated media containing the fusion
proteins were subjected to Western analysis using a monoclonal
anti-human MPR46 antibody for detection of the four fusion proteins (lanes 2-5). The amount of media was adopted to yield
signals of similar intensity for MPR46, assuming that the equal MPR46
signals reflect equal molarities of fusion proteins. PartB, the same amount of concentrated media as in A was subjected to
I-IGF-II cross-linking (lanes
2-5). As a control for IGF-II binding, 1 µg of
M6P/IGF-II receptor was analyzed (lane1). In the
experiment shown in B, the signal for fusion protein
10-15 cross-linked to
I-IGF-II (lane2) was lower than in other experiments. The apparent
molecular masses of proteins and cross-linked products are indicated.
In A and B, SDS-PAGE was performed using 10%
acrylamide.
The Single M6P/IGF-II Receptor Repeat 10 Fails to Bind
IGF-II
Repeat 10 was the only repeat that was spanned in its
full length by the peptides forming the 23-kDa IGF-II-binding
proteolytic fragment. To examine whether repeat 10 is sufficient to
bind IGF-II, fusion protein 10 was expressed. It consists of the
M6P/IGF-II receptor repeat 10 (amino acids 1367-1515) fused to
the extracytoplasmic domain of the MPR46 (see Fig. 6). By Western
analysis, fusion protein 10 has a mass of 51 kDa (Fig. 7A, lane5), which fits well to
the theoretical mass of 50 kDa. Analyzed in the IGF-II cross-linking
assay, no cross-linked product could be detected (Fig. 7B, lane5), even after
prolonged exposure of the x-ray film (data not shown).
M6P/IGF-II Receptor Repeats 10-11 Bind and
Cross-link IGF-II
The failure of fusion protein 10 to bind
IGF-II can be explained in two ways; the IGF-II-binding repeat may be
located carboxyl-terminally of repeat 10, or repeat 10 binds and
cross-links IGF-II only in the presence of the C-terminal repeats. To
determine whether the presence of repeat 11 is required for the binding
of IGF-II, we expressed the fusion protein 10-11, containing
repeats 10-11 (amino acids 1367-1650, see Fig. 6). In
Western analysis we detected a polypeptide with the mass of 65 kDa (Fig. 7A, lane4), which corresponds
to the theoretical mass. Cross-linked to IGF-II a product of 65 kDa was
detected (Fig. 7B, lane4), which
deviates from the calculated mass of 72.5 kDa. Half-maximal inhibition
of I-IGF-II cross-linking was estimated at about 200
nM unlabeled IGF-II (). These results indicate
that the presence of repeat 11 is essential for binding or
cross-linking of IGF-II.
M6P/IGF-II Receptor Repeat 11 Binds and Cross-links
IGF-II Independently of Repeat 10
To analyze, if repeat 11 binds
and cross-links IGF-II independent of repeat 10, we expressed the
fusion protein 11-15, which contains repeats 11-15 (amino
acids 1508-2293 of the M6P/IGF-II receptor, see Fig. 6).
The molecular mass of 125 kDa in the Western analysis fits well to the
theoretical mass of 122 kDa (Fig. 7A, lane3). Subjected to the IGF-II binding assay, a cross-linked
product of 130 kDa was detected, which corresponds to the calculated
mass (Fig. 7B, lane3). Half-maximal
inhibition of cross-linking was estimated at about 40 nM unlabeled IGF-II (). Additional cross-linked products
in the range of 10-45 kDa bind IGF-II with similar affinity,
indicating that they consist of fragments of fusion protein
11-15. Since this fragments failed to bind anti-MPR46 antibody in
Western analysis (Fig. 7A, lane3), we
suggest that they lack the MPR46 domain. These results clearly
demonstrate that repeat 11 is sufficient for binding of IGF-II.
I-IGF-II cross-linked peptides with
molecular masses of 14-18 kDa were isolated. N-terminal
sequencing revealed that the peptides extend from the C-terminal fourth
of repeat 10 to the N-terminal half of repeat 11 (corresponding to
amino acids 1479-1581 of the human M6P/IGF-II receptor). Since
lysine residues are missing in the C-terminal fourth of repeat 10, the
authors concluded that IGF-II was cross-linked to one or several of the
four lysine residues in the N-terminal half of repeat 11. Due to the
experimental approach, it could not be determined whether the isolated
fragments contain the IGF-II binding site, since the sites for binding
and cross-linking might reside in different receptor repeats. In view
of the results presented here, it is apparent that the cross-linking
sites in repeat 11 are close to the IGF-II binding site and that only
lysine residues 1509 and 1545 remain as candidate residues for
cross-linking.
I-IGF-II
cross-linking to fusion protein 10-15 corresponds to the value of
15 nM determined for the entire M6P/IGF-II receptor. The
values of 40 and 200 nM determined for the fusion proteins
11-15 and 10-11 indicate that the strength of binding
IGF-II is influenced by the presence of repeats 10 and 12-15.
Table: Characterization of the binding of IFG-II to
M6P/IGF-II receptor and the fusion proteins
I-IGF-II was
carried out in the absence and presence of 10
to
10
M unlabeled IGF-II as indicated. After
SDS-PAGE and autoradiography, cross-linked
I-IGF-II was
quantified by densitometry of the x-ray film and expressed as
percentage of
I-IGF-II bound in the absence of unlabeled
IGF-II. All values represent the mean and standard deviation derived
from 14-20 independent determinations.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.