The N-terminal Disulfide Linkages of Human Insulin-like Growth
Factor-binding Protein-6 (hIGFBP-6) and hIGFBP-1 Are Different as
Determined by Mass Spectrometry*
Gregory M.
Neumann
and
Leon A.
Bach§¶
From the
Department of Biochemistry, Latrobe
University, Bundoora, Victoria 3083 and the § Department of
Medicine, University of Melbourne, Austin & Repatriation Medical
Centre, Heidelberg, Victoria 3084, Australia
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ABSTRACT |
The actions of insulin-like growth factors (IGFs)
are modulated by a family of six high affinity binding proteins (IGFBPs 1-6). IGFBP-6 differs from other IGFBPs in having the highest affinity
for IGF-II and in binding IGF-I with 20-100-fold lower affinity.
IGFBPs 1-5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines. The complete disulfide linkages of
IGFBP-6 were determined using electrospray ionization mass
spectrometry of purified tryptic peptide complexes digested with
combinations of chymotrypsin, thermolysin, and endoproteinase Glu-C.
Numbering IGFBP-6 cysteines sequentially from the N terminus, the first
three disulfide linkages are Cys1-Cys2,
Cys3-Cys4, and
Cys5-Cys6. The next two linkages are
Cys7-Cys9 and
Cys8-Cys10, which are analogous to those
previously determined for IGFBP-3 and IGFBP-5. The C-terminal linkages
are Cys11-Cys12,
Cys13-Cys14, and
Cys15-Cys16, analogous to those previously
determined for IGFBP-2. Disulfide linkages of IGFBP-1 were partially
determined and show that Cys1 is not linked to
Cys2 and Cys3 is not linked to
Cys4. Analogous with IGFBP-3, IGFBP-5, and IGFBP-6,
Cys9-Cys11 and
Cys10-Cys12 of IGFBP-1 are also
disulfide-linked. The N-terminal linkages of IGFBP-6 differ
significantly from those of IGFBP-1 (and, by implication, the other
IGFBPs), which could contribute to the distinctive IGF binding
properties of IGFBP-6.
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INTRODUCTION |
The insulin-like growth factor
(IGF)1 system plays an
important role in normal physiology (1). Dysregulation of the system has also been implicated in many diseases including malignancy, atherosclerosis, and the development of diabetic complications. IGF-I
and IGF-II promote proliferation and differentiation of many cell
types. More recently, IGFs have been ascribed a potent role as
anti-apoptotic agents.
The actions of IGFs are modulated by a family of six structurally
related high affinity IGF-binding proteins (IGFBPs 1-6) (1-3). The N-
and C-terminal domains of IGFBPs 1-6 share significant sequence
homology, whereas the central regions linking these domains are not
homologous with each other. Under differing circumstances, IGFBPs may
inhibit or enhance IGF actions. The IGFBPs differ in their regulation,
relative IGF binding affinities, susceptibility to proteolysis by
specific proteases, and sites of synthesis. The IGFBPs therefore
constitute a flexible system for the regulation of IGF activity.
IGFBP-6 is an O-linked glycoprotein, which differs from
IGFBPs 1-5 in a number of significant respects. Of the IGFBPs,
IGFBP-6 has the highest binding affinity for IGF-II (4). IGFBP-6 also has the highest specificity for IGF-II, exhibiting a 20-100-fold higher binding affinity for IGF-II than IGF-I, whereas IGFBPs 1-5 bind
to IGF-II with equal or slightly higher affinity than IGF-I (4-7). A
potentially significant structural difference is that, whereas human
and rat IGFBPs 1-5 have 12 homologous N-terminal and 6 homologous
C-terminal cysteines, 2 of the N-terminal cysteines are not present in
human and rat IGFBP-6 and a short sequence containing another 2 N-terminal cysteines is missing from rat IGFBP-6 (8).
Disulfide linkages of cysteines are important for the correct folding
and maintenance of the three-dimensional structure of many proteins.
The N-terminal cysteines of the IGFBPs are disulfide-linked to each
other, as are the C-terminal cysteines, with no disulfide linkages
between the N- and C-terminal domains (2). To date, a complete set of
disulfide linkages has not been reported for any of the IGFBPs.
Recently, the three C-terminal disulfide linkages of bovine IGFBP-2
were determined (9). The C-terminal disulfide linkages of human IGFBP-6
were also partially determined (10) and are consistent with those of
IGFBP-2. We recently suggested that the N-terminal region of IGFBP-6
consists of two disulfide-linked domains, the first including the six
most N-terminal cysteines and the second including the next four
cysteines in the sequence (10). A recent structural study of IGFBP-5
has shown that the second of these domains contains the high affinity
IGF binding site (11); the two disulfide linkages within this domain
are the same as those determined for the homologous cysteines of
IGFBP-3 (12). The other N-terminal domain of IGFBP-5, containing the eight most N-terminal cysteines, is thought to form a separate domain
that does not interact with the high affinity binding site of IGFBP-5
nor strongly influence IGF binding to this site (11).
IGFBPs 1-5 contain a GCGCC motif in their N-terminal sequences, which
is replaced by GCAEA in human IGFBP-6 (8). Therefore, unless the two
adjacent cysteines in IGFBPs 1-5 are disulfide-linked, which is
unlikely on structural grounds, the disulfide linkages of IGFBP-6 in
this domain must differ from those of the other IGFBPs. A chimera of
the N-terminal and non-conserved middle regions of IGFBP-6 with the
C-terminal region of IGFBP-5 retains preferential binding affinity for
IGF-II over IGF-I (13). Since the non-conserved middle regions of
IGFBPs are not thought to be directly involved in high affinity IGF
binding, this suggests that the determinants of IGF-II binding
preference are located in the N-terminal region. In order to further
characterize the structural differences between IGFBP-6 and IGFBPs
1-5, we completely solved the disulfide linkages of human IGFBP-6
using electrospray ionization mass spectrometry (14) and reverse-phase
HPLC of proteolytic digests. Since none of the N-terminal disulfide
linkages and only a few other linkages of some of IGFBPs 1-5 are
known, we also partially solved the disulfide linkages of IGFBP-1
to provide a basis for comparison. We found that at least two of the
N-terminal disulfide linkages of IGFBP-1 are different from those of
IGFBP-6.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant Human
IGFBP-6--
Recombinant glycosylated human IGFBP-6 (hIGFBP-6) was
previously expressed in Chinese hamster ovary cells and purified by IGF-II affinity chromatography and reverse-phase FPLC, but was contaminated with endogenously synthesized IGFBP-4, which copurified with hIGFBP-6, necessitating lectin chromatography for removal of the
IGFBP-4 (10). For this reason, human embryonic kidney 293 cells, which
synthesize only small amounts of IGFBP-2 (15), were stably transfected
with phBP6-E3, the eukaryotic expression vector encoding human IGFBP-6
(16), using LipofectAMINE. Geneticin-resistant clones were selected,
and the colony producing the highest levels of hIGFBP-6 were recloned
and expanded for production of recombinant protein.
hIGFBP-6 was purified from conditioned medium by IGF-II affinity
chromatography followed by reverse-phase medium-pressure chromatography
(ProRPC 5/10 on an FPLC system, Amersham Pharmacia Biotech) using a
16-40% acetonitrile, 0.1% trifluoroacetic acid gradient (17). The
identity and purity of hIGFBP-6 were confirmed by N-terminal amino acid sequencing.
Purification of Human IGFBP-1--
Human IGFBP-1 (hIGFBP-1) was
purified from amniotic fluid by acid gel filtration (Bio-Gel P-30,
Bio-Rad), IGF-II affinity chromatography and reverse phase FPLC as
described previously (4). Identity and purity of hIGFBP-1 were
confirmed by immunoblotting and silver staining, respectively.
Proteolytic Digestion and Reverse-phase HPLC of
hIGFBP-6--
hIGFBP-6 (40 µg) was denatured without reduction (1 h,
37 °C) in 10 µl of 8 M urea, 100 mM
Tris-Cl (pH 7.5) and digested (20 h, 21 °C) with 1 µg of
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Worthington) in a total volume of 60 µl of 100 mM Tris-Cl (pH 7.5), 5 mM CaCl2,
1.3 M urea. This was followed by reverse-phase HPLC
fractionation as described previously (10), using a C18 column (1 mm × 250 mm), 40 µl/min flow rate, and a linear applied
gradient of 0-50% acetonitrile, 0.1% trifluoroacetic acid over 150 min. HPLC fractions containing disulfide-linked tryptic fragments were
identified by mass spectrometry (ESMS) as described below. Aliquots (50 pmol) were vacuum concentrated (SpeedVac) to 1-2 µl and further
digested (20 h, 21 °C) with 1-4 pmol of Staphylococcus
aureus V8 protease (endoproteinase Glu-C, Sigma),
thermolysin (Sigma),
N
-p-tosyl-L-lysine
chloromethyl ketone-treated chymotrypsin (Worthington), or both
thermolysin and chymotrypsin, in a total volume of 3 µl of 50 mM ammonium bicarbonate (pH 7.8), followed by further ESMS analysis.
Proteolytic Digestion and Reverse-phase HPLC of
hIGFBP-1--
Proteolytic digestion of hIGFBP-1 and reverse-phase HPLC
was carried out as described above for hIGFBP-6, except that 20 µg of
hIGFBP-1 was digested (20 h, 21 °C) with 0.5 µg of trypsin and 0.5 µg of S. aureus V8 protease (endoproteinase Glu-C/Asp-C when in phosphate buffer) combined in 45 µl of 50 mM
sodium phosphate (pH 7.8), 0.8 M urea. After ESMS analysis
of reverse-phase HPLC fractions, 5-pmol aliquots of the isolated 5-kDa
N-terminal hIGFBP-1 fragment were digested (20 h, 21 °C) with 1 pmol
of thermolysin in 2 µl of 100 mM ammonium bicarbonate (pH
7.8) and re-analyzed by ESMS.
Electrospray Ionization Mass Spectrometry (ESMS)--
Samples
(1-3 µl) of HPLC fractions and their digests were mixed with 3-6
µl of 50% acetonitrile, 0.1% formic acid prior to ESMS, which was
performed as described previously (10, 18) on a Perkin-Elmer Sciex
API-300 triple quadrupole mass spectrometer with micro-ion spray ion
source, calibrated to an accuracy of ±0.01% using singly charged
poly(propylene glycol) ions. MS/MS spectra (Q3 scans) were obtained
using nitrogen collision gas (4 millitorr pressure, 20.7 cm cell
length) and optimized collision energies of 32-64 eV. Signal-averaged
raw mass spectra were analyzed manually and transformed to a true mass
scale using the PE-Sciex BioMultiview program Biospec Reconstruct.
Peptide sequences inferred from observed masses were confirmed by
analysis of MS/MS spectra manually and using the BioMultiview programs
Predict Sequence and Peptide Fragments.
Amino Acid Sequencing--
N-terminal amino acid sequencing was
performed by sequential Edman degradation using a Hewlett-Packard
G1005A automated protein sequencing system, calibrated with
phenylthiohydantoin-amino acid standards prior to each sequencing run.
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RESULTS |
Expression and Purification of hIGFBP-6 in 293 Cells--
Approximately 300 µg of recombinant human IGFBP-6 was
purified from 500 ml of medium conditioned by stably transfected 293 human embryonic kidney cells. N-terminal Edman sequencing showed that
~80% of the protein commenced at Arg28 with the sequence
R(C)PG(C)GQG (numbering based on the sequence of the IGFBP-6 precursor
protein, SWISS-PROT accession number P24592; Fig.
1). The remainder of the protein
commenced at Leu26 with the sequence LAR(C)PG(C)G. This
heterogeneity of the N-terminal sequence is consistent with previous
studies of IGFBP-6 purified from natural and recombinant sources (10,
19, 20). The previously observed absence of Gly240 from
~70% of IGFBP-6 (10) was also identified in C-terminal fragments
analyzed in the present study. In the results that follow, cysteines
are numbered 1-16 in hIGFBP-6 and 1-18 in hIGFBP-1 sequentially from
the N terminus (Fig. 1).

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Fig. 1.
Amino acid sequences of hIGFBP-6 and
hIGFBP-1. Amino acids are numbered according to the SWISS-PROT
entries for proIGFBP-6 (accession number P24592) and proIGFBP-1
(accession number P08833).
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Purification of Disulfide-linked Tryptic Fragments of
hIGFBP-6--
Disulfide-linked tryptic fragments of hIGFBP-6 were
produced by tryptic digestion of 40 µg of purified hIGFBP-6
followed by C18 reverse-phase HPLC fractionation, which yielded a HPLC
profile (Fig. 2) similar to that
following a previous digestion using the same procedure on purified
hIGFBP-6 expressed in Chinese hamster ovary cells (10). ESMS analysis
of all major and some minor peak fractions confirmed that the three
most intense peaks (Fig. 2, peaks 1-3)
correspond to the same three hIGFBP-6 disulfide-linked tryptic
fragments of hIGFBP-6 that were previously identified by ESMS and Edman
sequencing (10) and that other major and minor peptide fragments were
also as previously identified. As reported previously (10), attempts to
release disulfide-linked peptides by reduction with 2-mercaptoethanol
in concentrations compatible with electrospray ionization (less than a
few percent, v/v) were unsuccessful at room temperature and resulted in
peptide degradation and/or loss at elevated temperatures. Therefore,
assigned sequences for all fragments with masses below 2300 Da were
confirmed by MS/MS fragmentation analysis as described under
"Experimental Procedures."

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Fig. 2.
Reverse-phase HPLC fractionation of a tryptic
digest of hIGFBP-6. As described under "Experimental
Procedures," hIGFBP-6 (40 µg) was denatured without reduction,
digested with trypsin, and subjected to C18 reverse-phase HPLC using a
gradient of 0-50% acetonitrile, 0.1% trifluoroacetic acid over 150 min. Upper trace, A215.
Lower trace, A280.
Peptides were identified by ESMS. Samples of fractions 1, 2, and 3, containing disulfide-linked tryptic peptide complexes, were further
analyzed by ESMS after digestion with chymotrypsin or thermolysin (or
both together), or S. aureus V8 protease (endoproteinase
Glu-C), as described in Table I.
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Fractions 1, 2, and 3 were further digested and reanalyzed by ESMS, as
described below. The peptide complexes in these three fractions account
for 14 of the 16 cysteines in hIGFBP-6. By default, and as previously
determined (10), the remaining two cysteines (Cys11 and
Cys12 in the sequences below) are disulfide-linked, and
connect glycosylated (140-165)NPGTSTTPSQPNSAGVQDTEMGPC11RR to
(181-193)GAQTLYVPNC12DHR. A number of glycoforms of this
complex, with masses corresponding to carbohydrate contents of 3-7
N-acetylhexosamine-hexose disaccharides and 0-5 sialic
acids, were identified as minor components of peak fraction 1 (Fig. 2)
and nearby fractions (data not shown), thereby confirming that
Cys11 is indeed linked to Cys12 in hIGFBP-6
expressed in 293 cells.
ESMS of Proteolytic Digests of hIGFBP-6 Disulfide-linked Tryptic
Fragments--
The three disulfide-linked tryptic complexes purified
as HPLC fractions 1, 2, and 3 (Fig. 2) were subjected to further
digestion by S. aureus V8 protease (endoproteinase Glu-C),
thermolysin, chymotrypsin, or a mixture of chymotrypsin and
thermolysin, as detailed in Table I.
Samples of fraction 2 (Fig. 2), containing the 3634-Da N-terminal
tryptic fragment
(28-65)RC1PGC2GQGVQAGC3PGGC4VEEEDGGSPAEGC5AEAEGC6LR
with three intramolecular disulfide linkages, were digested with
endoproteinase Glu-C and reanalyzed by ESMS (Table I). This revealed digestion into two complementary fragments with masses of 1619 and 2032 Da (Table I), as shown in Fig.
3A. Minor fragments were
present with extra or missing Glu (E), consistent with alternate Glu-C
cleavages in the sequence EEED49 (Fig. 3A), and
minor fragments due to additional cleavage at one or more Glu residues
(E) in the sequence EGCAEAE61 were also observed at low
mass (not shown). The identity of the 1619-Da fragment as
(49-65)DGGSPAEGC5AEAEGC6LR, with the
disulfide linkage Cys5-Cys6, was confirmed by
MS/MS collisional fragmentation (spectrum not shown) of the doubly
protonated (m/z 811) ion, which generated the following
b-type (N-terminal) and complementary y-type (C-terminal) sequence
ions: b2-b8, y9-y13
(singly charged), and y13-y15 (doubly charged). The ions b9-b14 and
y3-y8 were completely absent, as expected due
to the presence of the disulfide link bridging the cysteines at
positions 9 and 15 in the fragment sequence (21).
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Table I
ESMS analysis of hIGFBP-6 disulfide-linked proteolytic fragments and
deduced disulfide linkages
Molecular masses of major observed components were determined by ESMS
of disulfide-linked tryptic peptide complexes in HPLC fractions 1, 2, and 3 (Fig. 2), with or without additional digestion (as indicated
under "protease") by endoproteinase Glu-C (Glu-C), thermolysin
(Therm), chymotrypsin (Chym), or a mixture of chymotrypsin and
thermolysin (Ch&Th), as described under "Experimental Procedures."
Sequence assignments of tryptic peptide complexes were determined
previously by ESMS and Edman sequencing, as was the linkage
C11-C12 (10). Sequence assignments of fragments with
masses less than 2300 Da from additional digestion were confirmed by
MS/MS fragmentation analysis as in Fig. 3 (other data not shown). ESMS
(observed) masses averaged from several charge states (typically 2+ to
4+) are compared with calculated average molecular masses above 2000 Da, but with monoisotopic (m) masses below 2000 Da due to the lower
observed charge states (1+ to 2+). Uncertainties are mean deviations
plus calibration uncertainty. Calculated masses assume disulfide
linkage of all cysteines (C), which are numbered 1-16 sequentially
(Fig. 1). Long sequences are abbreviated ( ). Deduced disulfide
linkages are summarized at the foot of the table and shown in Fig. 4.
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Fig. 3.
Electrospray ionization mass spectra of an
endoproteinase Glu-C digest of the 3.6-kDa N-terminal tryptic fragment
of hIGFBP-6. Aliquots (50 pmol) of fraction 2 (Fig. 2), containing
the purified 3634-Da N-terminal tryptic fragment
RCPGCGQGVQAGCPGGCVEEEDGGSPAEGCAEAEGCLR with three intramolecular
disulfide linkages, were digested with endoproteinase Glu-C and
analyzed by ESMS as described under "Experimental Procedures."
Upper spectrum (A), mass spectrum
(transform) showing two major peptide fragments with deduced disulfide
linkages (see below) as indicated. Lower spectrum
(B), MS/MS fragmentation spectrum (48-eV collision energy)
of the [M+2H]2+ ion (m/z 1017) of the
RCPGCGQGVQAGCPGGCVEEE fragment (with two intramolecular disulfide
linkages) shown in spectrum A. As indicated
schematically, the two disulfide linkages are sequential (do not cross
over), as determined from the presence of prominent b-type (N-terminal)
fragment ions b5-b12 and supported by the
presence of b17-b21, but absence of
b13-b16 and b2-b4.
Above m/z 1300 (not shown), only weak signals due to singly
charged b17-b19 fragment ions were
observed.
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The identity of the 2031-Da fragment as
(28-48)RC1PGC2GQGVQAGC3PGGC4VEEE,
with two intramolecular disulfide linkages, was confirmed and the
disulfide linkage arrangement also determined by MS/MS collisional
fragmentation of the doubly protonated (m/z 1017) ion (Fig.
3B). Prominent b-type (N-terminal) fragment ions
b5-b12 and b17-b21
were observed, while the ions b13-b16 and
b2-b4 were conspicuously absent, as expected
due to the presence of bridging disulfide linkages (21). The key
observation is the presence of prominent
b5-b12 ions, which would not be observed
unless the two disulide linkages are sequential (do not cross
over), as shown schematically in Fig. 3B. The linkage
arrangement Cys1-Cys2 was independently
determined by ESMS analysis of thermolysin digests of the 3634-Da
N-terminal tryptic fragment, which were found to contain the
complementary fragments (28-35)RC1PGC2GQG and
(36-65)VQAGC3PGGC4VEEEDGGSPAEGC5AEAEGC6LR
as major components (Table I). Combining this result with that from
Glu-C digestion (Fig. 3A) independently implies the same
disulfide linkages as those determined by MS/MS (Fig. 3B). It is concluded that the three most N-terminal disulfide linkages of
hIGFBP-6 are Cys1-Cys2,
Cys3-Cys4, and
Cys5-Cys6.
Samples of fraction 3 (Fig. 2), containing the 3680-Da tryptic complex
(67-95)EGQEC7GVYTPNC8APGLQC9HPPKDDEAPLR
disulfide-linked to (104-108)C10LPAR (Table I), were
analyzed by ESMS after digestion with thermolysin, chymotrypsin, or a
mixture of thermolysin and chymotrypsin. Digestion of the fraction 3 complex with chymotrypsin or thermolysin alone resulted in an 18-Da
mass increase of the complex (or with thermolysin, the complex minus
C-terminal LR), consistent with peptide bond hydrolysis at V-Y (or G-L
with thermolysin) but with fragments still disulfide-linked (data not
shown). Digestion of the complex with a mixture of thermolysin and
chymotrypsin resulted in the generation of two complementary fragments
with masses of 2231 and 1215 Da, corresponding to
(67-74)EGQEC7GVY linked to
(82-93)LQC9HPPKDDEAP and (75-81)TPNC8APG
linked to (104-108)C10LPAR, respectively (Table I). These
and other assignments were confirmed by MS/MS analysis (data not
shown). It is concluded that Cys7 is linked to
Cys9 and Cys8 is linked to
Cys10.
Samples of fraction 1 (Fig. 2) containing the C-terminal
disulfide-linked tryptic complex (200-202)QC13R linked to
(209-217)RGPC14WC15VDR, which is in turn
linked to (221-239)SLPGSPDGNGSSSC16PTGSS (Table I), were
digested with chymotrypsin at protease-to-peptide molar ratios that
were slightly (2-3-fold) higher than those used with other digests in
order to obtain complete digestion. ESMS analysis showed that two major
fragments with masses of 1021 and 2183 Da were generated (Table I);
these correspond to (200-202)QC13R linked to
(209-213)RGPC14W and (214-217)C15VDR
linked to (221-239)SLPGSPDGNGSSSC16PTGSS, as confirmed by
MS/MS analysis (data not shown). These results clearly differentiate
between the two possible linkage arrangements within the C-terminal
tryptic complex, indicating that the disulfide linkages are
Cys13-Cys14 and
Cys15-Cys16. Since Cys11 is
connected to Cys12 (Table I), all three C-terminal
disulfide linkages are therefore sequential. The complete disulfide
linkages determined for hIGFBP-6 are summarized at the foot of Table I
and shown in Fig. 4.

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Fig. 4.
Structural features of hIGFBP-6.
Numbers indicate cysteines involved in disulfide linkages
(numbering based on the proIGFBP-6 sequence, SWISS-PROT accession
number P24592, Fig. 1). The conserved N-terminal and C-terminal regions
of human IGFBP-6 are shaded, and the non-conserved
mid-region is unshaded. Cysteines are indicated by
vertical lines and disulfide linkages by
horizontal lines. Cysteines contained in the
sequence AGCPGGCVE, which is not present in the rat IGFBP-6 sequence
(8), are indicated by crosses. Previously determined
O-glycosylation sites (10) are indicated by
arrows.
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ESMS and Edman Sequencing of Proteolytic Fragments of
hIGFBP-1--
hIGFBP-1 was digested with a mixture of trypsin and
S. aureus V8 protease and subjected to C18 reverse-phase
HPLC fractionation as shown in Fig. 5.
All major and most minor HPLC peak fractions (Fig. 5) were analyzed by
ESMS (Table II) and initially identified by comparison of observed masses with those expected on the basis of
the hIGFBP-1 sequence (Fig. 1) and the protease specificities of
trypsin (Arg-C/Lys-C) and S. aureus V8 protease (Glu-C/Asp-C under the digest conditions employed), taking into account potential missed cleavages and possible disulfide linkage combinations. Use was
also made of UV absorbance at 280 nm compared with that at 215 nm (Fig.
5) to gauge the fractional content of tryptophan and tyrosine in
peptide fragments. Assignments of all peptides and disulfide-linked
peptide complexes with masses below 2300 Da (Table II) were
subsequently confirmed by MS/MS fragmentation analysis (data not
shown).

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Fig. 5.
Reverse-phase HPLC fractionation of a
proteolytic digest of hIGFBP-1. hIGFBP-1 (20 µg) was digested
with a mixture of trypsin and S. aureus V8 protease
(endoproteinase Glu-C/Asp-C) as described under "Experimental
Procedures" and subjected to C18 reverse-phase HPLC as in Fig. 2.
Upper trace, A215.
Lower trace, A280.
Peptides were identified by ESMS (Table II, Fig. 6) and the 5275-Da
N-terminal disulfide-linked peptide complex in peak fraction 16 additionally analyzed by Edman sequencing (Table III).
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Table II
ESMS analysis of hIGFBP-1 proteolytic fragments and deduced disulfide
linkages
HPLC fractions (as defined in Fig. 5) from reverse-phase HPLC of
hIGFBP-1 digested with a mixture of trypsin and S. aureus V8
protease (endoproteinase Glu-C/Asp-C) were analyzed by ESMS as
described under "Experimental Procedures," yielding observed masses
as indicated. Uncertainties are mean deviations plus calibration
uncertainty. Calculated molecular masses based on the hIGFBP-1 sequence
(Fig. 1) are monoisotopic below 2000 Da and average (av) above 2000 Da.
Assigned sequences for all fragments with masses below 2300 Da were
confirmed by MS/MS fragmentation analysis (data not shown) as in Fig.
3. The sequence assignment for the 5275-Da fraction 16 fragment (see
Fig. 6 for mass spectrum) was confirmed by Edman sequencing (Table
III). Disulfide-linked peptides (indicated by *) identified in HPLC
fractions 4, 13, 14, and 16 (indicated in bold) account for all
cysteines except C15, C16, C17, and
C18. Digestion of the 5275-Da fraction 16 complex by
thermolysin (Th) generated a major 3771-Da fragment (as indicated),
which corresponds to removal of (42-52)LGAAC9GVATAR linked to
(56-60)GLSC11R, demonstrating that C9 is connected to
C11. Deduced disulfide linkage information for hIGFBP-1 is
summarized at the foot of the table. All cysteines (C) are numbered
from 1 to 18 sequentially (Fig. 1).
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Major disulfide-linked peptide fragments were identified in HPLC
peak fractions 4, 13, 14, and 16 (Table II). Fraction 4 contained the fragment (53-55)Cys10AR linked to
(75-82) GQGACys12VQE, demonstrating that
Cys10 is linked to Cys12. Fraction 13 contained
the fragment (146-154)KWKEPCys13RIE linked to
(176-183)FYLPNCys14NK, demonstrating that
Cys13 is linked to Cys14. A related fragment
with the same disulfide linkage, but in which K146 had been
removed during digestion, was identified in Fraction 14, and additional
related fragments due to further removal of WK148 and/or
IE154 were identified as minor components of fractions 10 and 11 (data not shown).
Fraction 16 contained a 5275-Da fragment (Fig.
6), the mass of which corresponds
precisely with that calculated for the N-terminal disulfide-linked
complex consisting of the following fragments: (1-12)APWQC1APC2SAEK,
(13-25)LALC3PPVSASC4SE,
(29-52)SAGC5GC6C7PMC8ALPLGAAC9GVATAR,
and (56-60)GLSC11R (Table II, fragments are those
predicted for cleavage after R or K by trypsin, and after E by
endoproteinase Glu-C). This assignment was confirmed by N-terminal
Edman sequencing (Table III), the results
of which are consistent with the apparent purity of fraction 16 when
analyzed by ESMS (Fig. 6). Samples of fraction 16 were digested with
thermolysin and re-analyzed by ESMS, which revealed an additional major
3771-Da fragment (Table II) that was not present before digestion (Fig.
6) and was not present in a zero digestion-time control sample
(data not shown). This fragment corresponds to removal of
(42-52)LGAAC9GVATAR linked to (56-60)GLSC11R,
thereby demonstrating that Cys9 is linked to
Cys11. Fragments corresponding to the removed
disulfide-linked section (or products of its further digestion by
thermolysin on the N-terminal side of L, V, or A) may have been present
in digests but were not clearly identified. Further digestion of the
5275-Da N-terminal complex to determine the remaining four disulfide
linkages was precluded by the lack of suitable cleavage sites.

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Fig. 6.
Electrospray ionization mass spectrum of the
N-terminal disulfide-linked fragment of hIGFBP-1. Fraction 16 from
reverse-phase HPLC of proteolyzed hIGFBP-1 (Fig. 5) was analyzed by
ESMS as described under "Experimental Procedures." Upper
spectrum, unprocessed mass spectrum from 2 pmol of peptide
(in 2 µl of 50% acetonitrile, 0.1% formic acid), showing ions with
(left to right) 6, 5, 4, and 3 positive charges.
Lower spectrum, mass transform from
m/z to a true mass scale, showing the single major 5275-Da
N-terminal disulfide-linked fragment (identified in Tables II and III)
and adjacent minor peaks corresponding to oxygen, sodium, potassium,
and ferrous ion adducts.
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Table III
Edman sequencing of 5-kDa N-terminal disulfide-linked fragment of
hIGFBP-1
Results of Edman sequencing of the 5275-Da N-terminal disulfide-linked
peptide complex (HPLC fraction 16, Fig. 5) isolated as described in
Fig. 5 by reverse-phase HPLC of hIGFBP-1 digested with trypsin and
S. aureus V8 protease. The 5275-Da complex was initially
identified as consisting of the four disulfide-linked peptides shown in
Table II on the basis of the hIGFBP-1 sequence (Fig. 1), the
specificities of trypsin (Arg-C/Lys-C) and S. aureus V8
protease (Glu-C/Asp-C), and the ESMS mass (Table II). Corresponding
amino acid signals expected in each Edman cycle are compared with the
observed signals from 16 cycles of Edman sequencing carried out as
described under "Experimental Procedures." Multiple signals in
respective Edman cycles are arranged vertically according to the
assigned sequences, therefore some expected or observed signals are
repeated for ease of comparison. Dashes (-) represent cysteine (not
expected to be observed under Edman sequencing conditions) or expected
amino acid signal changes that were not observed. Observed Edman
signals that are underlined were equivocal due to carryover of the same
signal from a previous cycle, C-terminal amino acid washout, or weak
signals from serine or in cycles following proline. The Edman
sequencing data confirm the ESMS-based identification (Table II).
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The 5275-Da N-terminal disulfide-linked complex incorporates the
fragments (1-12)APWQC1APC2SAEK and
(13-25)LALC3PPVSASC4SE (Tables II and III),
each of which contains only two cysteines; therefore, in order to be
part of the 5275-Da complex, Cys1 cannot be connected to
Cys2 and Cys3 cannot be connected to
Cys4. For the same reason, Cys1,
Cys2, Cys3, and Cys4 cannot all be
interconnected. Given the result from thermolysin digestion of the
5275-Da complex (Cys9 linked to Cys11), it is
therefore concluded that one or both of Cys1 and
Cys2, and one or both of Cys3 and
Cys4 must be connected to two or four of the cysteines in
the sequence (29-41)SAGC5GC6C7PMC8ALP
(Table II).
The identified disulfide-linked peptides (Table II) account for all
hIGFBP-1 cysteines except the four C-terminal cysteines, Cys15 to Cys18. No peptide complexes containing
any of these cysteines were identified and, in particular, the single
complex expected to contain all four cysteines (Table II) was not
observed. All other expected fragments containing more than four amino
acids were recovered (Table II), and all fragments in all major and
minor peak fractions analyzed were successfully identified. For
example, fractions 10 and 15 (not listed in Table II) contained
peptides that were identified as oxidized (methionine sulfoxide) forms of those in fractions 12 and 16, respectively (respective masses 16 Da
higher; data not shown). The C-terminal peptides unaccounted for are:
(191-193)QC15E,
(200-212)AGLC16WC17VYPWNGK, and
(222-234)GDPNC18QIYFNVQN. While it is possible that these
are not linked to form a single complex, the loss of a single large
peptide complex (possibly due to excessive hydrophobicity or other
factors leading to aggregation) is more probable than the
simultaneous loss of two or more smaller entities. Therefore it is
concluded that Cys15 is most likely linked to
Cys16 or Cys17, and Cys18 is most
likely linked to Cys17 or Cys16. Deduced
disulfide linkage information for hIGFBP-1 is summarized at the foot of
Table II.
 |
DISCUSSION |
The IGFBPs are important modulators of IGF actions (1-3).
Although the actions of IGFBPs have been the subject of intense scrutiny for over a decade, only limited information regarding their
structure is available. The results of this study demonstrate for the
first time the complete disulfide linkages of an IGFBP. In addition,
the disulfide linkages of IGFBP-1 have been partially characterized and
compared with those of IGFBP-6, revealing that the N-terminal
disulfide linkages of these IGFBPs differ significantly. In contrast,
the C-terminal disulfide linkages of bovine IGFBP-2 (9) and human
IGFBP-6 are identical, and the disulfide linkages of IGFBP-5 within its
putative high affinity IGF binding domain (11) are identical to those
in the corresponding domains of IGFBP-6, IGFBP-1 (both shown in the
present study) and IGFBP-3 (12).
A distinctive feature of IGFBP-6 compared with IGFBPs 1-5 is its high
binding affinity for IGF-II and its marked preferential binding of
IGF-II relative to IGF-I. A chimera of the N-terminal and middle
regions of IGFBP-6 with the C-terminal region of IGFBP-5 retains the
binding preference for IGF-II (13). Since the non-conserved middle
regions of the IGFBPs are not thought to be directly involved in IGF
binding, the structural determinants of the IGF-II binding preference
are therefore likely to reside in the N terminus. It may be of
structural significance that rat IGFBP-6 shares the IGF-II binding
preference with human IGFBP-6 (22) despite lacking the
AGC3PGGC4VE peptide segment present in human
IGFBP-6 (8). Notably, the two N-terminal cysteines (Cys3
and Cys4) within this segment are disulfide-linked in human
IGFBP-6. It is therefore likely that the other N-terminal linkages in
rat IGFBP-6 are the same as those in human IGFBP-6.
The N-terminal disulfide linkages of IGFBP-6 differ substantially from
those of IGFBP-1, and, by implication, the other IGFBPs. The N-terminal
disulfide linkages of IGFBP-6 are the sequential linkages
Cys1-Cys2, Cys3-Cys4,
and Cys5-Cys6, whereas in IGFBP-1,
Cys1 is not linked to Cys2 and Cys3
is not linked to Cys4. Although these observations suggest
that a different local fold in the N-terminal region is likely, it is
possible that this is not the case. For example, the presence of
proline-containing sequences in the N-terminal regions of IGFBPs 1-5
could result in a similar fold to that stabilized by the disulfide
linkages in IGFBP-6.
Human and rat IGFBP-6 lack the adjacent cysteine (CC) pair that is part
of the GC5GC6C7 motif present in
the N-terminal domains of IGFBPs 1-5 (8). Although it is possible that
these two adjacent cysteines in IGFBPs 1-5 are disulfide-linked, this
is unlikely since disulfide linkage of adjacent cysteines requires a
relatively rare cis peptide bond between the cysteines (23).
Additionally, linkage of these adjacent cysteines would serve no
obvious structural purpose, which seems inconsistent with their being
conserved in all IGFBPs other than IGFBP-6. It is therefore probable
that these cysteines are disulfide-linked to other N-terminal cysteines
in IGFBPs 1-5.
While further investigation is clearly needed to determine the
N-terminal disulfide linkages of IGFBP-1 (and IGFBPs 2-5), it is
unlikely that this can be accomplished by peptide mapping techniques
relying on proteolysis alone, due to lack of suitable cleavage sites in
the conserved sequence C5GC6C7 and
elsewhere. Indeed, the N-terminal disulfide linkages of IGFBP-3 could not be determined by this approach (12). Appropriate cleavage sites could conceivably be introduced by site-directed mutagenesis. Peptide mapping methods could also be coupled with selective
mutagenesis of cysteines in order to determine disulfide linkages one
at a time, provided correct formation of remaining disulfide linkages was not affected.
The high affinity IGF binding site of IGFBP-5 has recently been
localized to the sequence containing Cys9 to
Cys12 of that protein (equivalent to Cys7 to
Cys10 of IGFBP-6) (11). Since IGFBPs -1, -3, and -6 share
the same disulfide linkages in this region as IGFBP-5 and there is
substantial homology between the IGFBPs of critical hydrophobic amino
acids involved in IGF binding, it is likely that this region confers high IGF binding affinity to all IGFBPs. It was suggested that the
N-terminal domain of IGFBP-5 does not significantly interact with the
high affinity binding site of IGFBP-5 nor influence IGF binding by this
site (11). With IGFBP-6, however, the substantially different disulfide
linkages of the N-terminal domain compared with those of IGFBP-1 (and
presumably the other IGFBPs) could result in the stabilization of a
different N-terminal domain structure which may be responsible for or
contribute to the distinctive IGF binding characteristics of IGFBP-6.
An alternate possibility is that the structures of the N-terminal
domains of IGFBP-6 and other IGFBPs are folded in a similar way that is
stabilized by quite different disulfide linkages. In that case,
assuming the remainder of the structure also to be similar in IGFBPs
1-6, the IGF-II binding preference of IGFBP-6 could depend more on
differences in the primary sequence compared with other IGFBPs than to
differences in three dimensional structure, although both are likely to
be influential.
It has been claimed that the C-terminal domain of the IGFBPs is
also involved in IGF binding (11, 24). Although many studies have
suggested that the C-terminal domains of IGFBPs do not independently bind IGFs (11, 25, 26), the presence of this domain substantially increases IGF binding affinity, possibly by stabilizing the IGF·IGFBP complex once the IGF has bound. In contrast, two studies have shown
that C-terminal fragments of IGFBP-2 bind IGFs with low affinity (27,
28). Interestingly, deletion of the sequence containing the two
C-terminal disulfide linkages eliminated the 12-fold IGF-II binding
preference of bovine IGFBP-2 over IGF-I (9). IGFBP-6 and IGFBP-2 share
a preference for IGF-II and have the same three C-terminal disulfide
linkages. It has not been demonstrated that these linkages are
different in the other IGFBPs, which lack substantial IGF-II binding
preference; but, if present, such a difference in the C-terminal
disulfide linkages (such as those connected to the conserved CWCV
sequence) could conceivably influence IGF-II binding specificity.
However, the sequences of IGFBPs 1-6 are homologous with respect to
C-terminal cysteines, suggesting that their disulfide linkages are
likely to be the same. Unfortunately, the peptide complex containing the four C-terminal cysteines of IGFBP-1, which would provide considerable information on this matter, was not recovered in the
present study. In any case, it would be difficult to reconcile the
IGF-II binding preference of the IGFBP-6/IGFBP-5 chimera mentioned above (13) with a C-terminal determinant of IGF-II binding preference.
In conclusion, we have determined all of the disulfide linkages of
human IGFBP-6. As suggested previously (10), the N-terminal linkages
fall into two domains comprising Cys1 to Cys6
(the N-terminal domain) and Cys7 to Cys10 (the
IGF binding domain), respectively. The six C-terminal cysteines of
IGFBP-6 form three disulfide bonds, and there are no linkages between
the N- and C-terminal regions. The N-terminal linkages differ
significantly from those of IGFBP-1 and therefore, by inference, those
of the other IGFBPs. In contrast, the remaining disulfide linkages of
IGFBP-6 are the same as those reported in other IGFBPs; all of these
linkages are therefore likely to be identical in IGFBPs 1-6. Knowledge
of the disulfide linkages of IGFBP-6 will facilitate further structural
studies of this protein as well as aiding in the rational design of
mutants for structure-function correlations.
 |
ACKNOWLEDGEMENTS |
We thank Joe Marinaro for assistance in
expression and purification of IGFBP-6 and Rosemary Condron for
performing Edman N-terminal sequencing.
 |
FOOTNOTES |
*
This work was supported by grants from the Austin Hospital
Medical Research Foundation and the Sir Edward Dunlop Medical Research Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Medicine,
University of Melbourne, Austin & Repatriation Medical Centre (Austin
Campus), Heidelberg, Victoria 3084, Australia. Tel.: 61-3-9496-3581; Fax: 61-3-9457-5485; E-mail: bach{at}austin.unimelb.edu.au.
 |
ABBREVIATIONS |
The abbreviations used are:
IGF, insulin-like
growth factor;
IGFBP, insulin-like growth factor-binding protein;
hIGFBP, human insulin-like growth factor-binding protein;
FPLC, fast
protein liquid chromatography;
HPLC, high pressure liquid
chromatography;
ESMS, electrospray ionization mass spectrometry;
MS/MS, mass spectrometry/mass spectrometry.
 |
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