(Received for publication, June 24, 1996, and in revised form, October 1, 1996)
From the While previous studies revealed that matrix
vesicles (MV) contain a nucleational core (NC) that converts to apatite
when incubated with synthetic cartilage lymph, the initial mineral
phase present in MV is not well characterized. This study explored the
physicochemical nature of this Ca2+ and Pi-rich
NC. MV, isolated from growth plate cartilage, were analyzed directly by
solid-state 31P NMR, or incubated with hydrazine or NaOCl
to remove organic constituents. Other samples of MV were subjected to
sequential treatments with enzymes, salt solutions, and detergents to
expose the NC. We examined the NC using transmission electron
microscopy, energy-dispersive analysis with x-rays, and electron and
x-ray diffraction, Fourier transform-infrared spectroscopy, high
performance thin-layer chromatographic analysis, and SDS-polyacrylamide
gel electrophoresis. We found that most of the MV proteins and lipids could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis showed
that annexin V, a phosphatidylserine (PS)-dependent
Ca2+-binding protein, was the major protein in the NC; high
performance thin-layer chromatographic analysis revealed that the
detergents removed the majority of the polar lipids, but left
significant free cholesterol and fatty acids, and small but critical
amounts of PS. Transmission electron microscopy showed that the NC was composed of clusters of ~1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and Pi with a
Ca/P ratio of 1.06 ± 0.01. Electron diffraction, x-ray
diffraction, and Fourier transform-infrared analysis all indicated that
the NC was noncrystalline. 1H-Cross-polarization
31P NMR indicated that the solid phase of MV was an
HPO42 De novo initiation and propagation of calcium phosphate
mineral formation in vertebrate bone formation is a multistep process. In growth plate cartilage, much evidence indicates that extracellular microstructures known as matrix vesicles
(MV)1 play a key role in the induction of
the first solid phase mineral (1-3). MV are produced by budding from
the plasma membrane into sites of intended calcification by cellularly
controlled mechanisms (4-6). Previous biochemical (7-9) and
ultrastructural (10, 11) studies have shown that MV contain large
amounts of calcium and phosphate; however, the initial mineral phase
has not yet been characterized. X-ray diffraction (12), and FT-IR (13) and FT-Raman (14) spectroscopic analyses all indicate that the initial
mineral form in MV is noncrystalline.
In previous work, we showed that it was possible to strip away
nonessential components from the MV using various chemical and
detergent extractions leaving a residual core that retained the ability
to induce mineral formation (15). The activity of this detergent-stable
nucleational core (NC) was inhibited by Zn2+ and was
destroyed by treatment with pH 6 citrate buffer. This acid lability is
similar to that of synthetic amorphous calcium phosphate (ACP) (15,
16). Further, in our assay system, the calcification of both MV and ACP
share the same pH-dependent kinetic profile when incubated
in synthetic cartilage lymph (SCL) (17). Based on these observations,
the NC of MV appears to contain ACP-like mineral; however, both FT-IR
and FT-Raman spectroscopy failed to detect classical ACP in native MV
(13, 14).
Several other forms of calcium phosphate, i.e. dicalcium
phosphate dihydrate (brushite) and octacalcium phosphate (OCP), have also been postulated to be precursors to hydroxyapatite (HAP) in
vivo (18). By FT-IR and FT-Raman analyses (13, 14), the first
crystalline mineral phase formed by MV incubated in metastable SCL was
OCP-like in character. After 24 h it appears to be poorly crystalline HAP, similar to that present in bone (19, 20).
Lipids are present in the extracellular matrix at sites of
calcification (21), and some are associated with the mineral phase
(22). Varying proportions of the predominant lipid associated with the
mineral, PS, are complexed with Ca2+ and Pi
(22-24). These Ca2+-PS-Pi complexes (CPLX) are
present in bone, cartilage, and other mineralized tissues (25) and are
especially enriched in MV (26). In vitro experiments have
shown that both CPLX and ACP, when incubated in synthetic lymphs, can
lead to formation of HAP (18, 27, 28). In these studies we sought to
determine unequivocally whether CPLX and ACP are present in the NC of
MV. Establishment of the physicochemical properties of the nucleational
core (the first mineral phase) would provide an important clue to how
MVs are formed and how they function as the primary nucleator in growth plate cartilage calcification.
In this investigation we examined the physicochemical properties of the
residual core material in MV remaining after sequential treatments with
enzyme, buffered salt, and detergent solutions. We also used treatment
with hydrazine or hypochlorite to remove organic matter. We assayed the
kinetics of mineral phase formation after each treatment procedure to
establish that nucleational activity had been retained. Focus was then
placed on structural characterization of the residue (i.e.
nucleational core, NC) using the following physicochemical methods:
transmission electron microscopy (TEM) for structure, energy-dispersive
analysis with x-rays (EDAX) and chemical analyses for elemental
composition, electron and x-ray diffraction for crystallinity, and
FT-IR and solid-state 31P NMR spectroscopy for chemical
structure. High performance thin-layer chromatography (HPTLC) was used
to analyze lipids; SDS-PAGE was used to analyze proteins.
Collagenase-released matrix
vesicles (CRMV) were isolated from growth plate cartilage of
6-8-week-old broiler strain chickens as described previously (15).
Briefly, tissues were digested with 0.1% trypsin (type III, Sigma) at
37 °C for 20 min in synthetic cartilage lymph (SCL). SCL contained 2 mM Ca2+ and 1.42 mM Pi,
in addition to 104.5 mM Na+, 133.5 mM Cl Hyaluronidase-released matrix vesicles were isolated from the same
tissues by incubating in SCL containing 0.25% hyaluronidase (Type IS,
Sigma) at 37 °C for 90 min, followed by incubation in SCL containing
collagenase (500 units/g of tissue) at 37 °C for 3 h (30).
After vortexing, hyaluronidase-released matrix vesicles were isolated
by differential centrifugation as before. The MV pellets were
resuspended in SCL containing 1 mM Ca2+ .
CRMV were extracted
sequentially with hyaluronidase, water, pure collagenase, high salt,
and then with detergent, to expose the acid-labile NC (Fig. 1). While
some of these procedures had been explored individually before to
determine their effects on MV mineralization (15), in this study these
procedures were used sequentially to fully strip away the non-essential
components. The final pellet was resuspended in SCL containing 1.0 mM Ca2+.
CRMV (1.0 mg of Lowry protein),
and the sequentially treated residual cores, were decalcified with 1 M EDTA, pH 7.5, for 30 min at 25 °C, dialyzed against
deionized water overnight at 4 °C with constant stirring, and then
lyophilized. The demineralized residue was extracted sequentially with
chloroform-methanol (2:1, v/v) (Extract 1) and
chloroform-methanol-concentrated HCl (200:100:1, v/v/v) (Extract 2) as
described previously (22). The dried crude lipids from Extracts 1 and 2 were partitioned through a micro-Sephadex G-25 column to remove
nonlipid contaminants (31). The pure lipids were then analyzed by HPTLC
on Whatman LHP-K silica gel plates as described previously (15, 32).
Mixtures containing 10 µg each of the various standard lipids were
applied to separate lanes on the same plate as the lipid samples.
A multiwell microplate mineralization
assay based on light scattering (33) was used for real-time measurement
of mineral formation by MV, and their subfractions. Samples were
prepared for mineralization assays as follows. Aliquots of the MV stock solution were diluted 100-fold to a final concentration of 50 µg of
protein/ml of SCL containing 2.0 mM Ca2+;
nucleational core pellets (Fig. 1, Pellet F) were
resuspended SCL to a final concentration of 50 µg of protein/ml,
based on the initial protein concentration (Fig. 1,
Pellet A). For kinetic assays of mineral formation, 160-µl
aliquots of the diluted vesicle or nucleational core suspensions were
placed into 96-well half-area tissue culture clusters (Costar,
Cambridge, MA) and incubated at 37 °C. An IEMS Reader MF instrument
(Labsystems, Needham Heights, MA) was used to take absorbency readings
at 340 nm at 15-min intervals.
MV and their subfractions were digested in 0.1 N
HCl at 25 °C overnight. The digests were clarified by centrifugation
using a Beckman Airfuge (22 p.s.i., 8 min) and then samples were
removed for mineral ion analysis. Ca2+ was analyzed by the
method of Baginsky et al. (34); Pi was determined by the method of Ames (35). For total P analysis, a small
volume of 70% HClO4 containing 0.01% ammonium molybdate (36) was added to the dry samples, which were digested at 140 °C for
15 min or longer until the samples were completely clear. Then aliquots
of the digestate were taken for Pi analysis as above.
SDS-PAGE was used to analyze the
protein composition of the various MV fractions. A 3.0% stacking gel
and a 7.5-15% gradient resolving gel were used, following the method
of Laemmli (37). Gels were stained with Coomassie Brilliant Blue R-250.
For FT-IR analyses, lyophilized samples of the NC (~1
mg) were incorporated into KBr (~300 mg) and pressed into a pellet
under vacuum at 12,000 psi pressure, and then examined over a range from 4000 to 400 cm MV subfractions were
lyophilized and samples (<1 mg) dispersed in 100% ethanol by
sonication. One drop of the suspension was added to one drop of
deionized H2O, and transferred to carbon-coated grids,
blotted, and dried. The grids were viewed using an Hitachi H-8000 TEM
at 200 keV accelerating voltage. Grids were not stained to
increase contrast. For EDAX analyses the following parameters were
used: accelerating voltage, 25 keV; angle of incidence beam, 90°;
x-ray emergence angle, 41.8°; x-ray window incidence angle, 6.8°;
counting time, 200 s. At least five representative areas were
analyzed for elemental composition. Values were calculated in atomic
percent normalized to 100 after eight iterations. Areas shown to be
rich in Ca and P by EDAX analysis were analyzed by electron diffraction
for the presence of crystalline mineral.
Each MV subfraction was
analyzed by x-ray diffraction for the presence of crystalline solids
before and after incubation in SCL using a Phillips Norelco x-ray
diffractometer using CuK Lyophilized CRMV were treated
with hydrazine monohydrate (Aldrich, 98%, 166 µl/mg of protein) at
60 °C for 24 h (40). The mixture was centrifuged using a
Beckman Airfuge at 22 p.s.i. for 8 min and the insoluble residue
washed twice with 100% ethanol. The residue was 1) dried under vacuum
before FT-IR analysis, 2) dispersed in 100% ethanol for TEM analysis,
or 3) dispersed in SCL containing 1 mM Ca2+
before measuring Ca2+ uptake.
Vesicle samples containing 50 µg of protein were treated
with a solution of either NaOCl, pH 12 (50 or 100 mM final
concentration), or NaOH, pH 12 and 13 (10 or 100 mM final
concentration) in a total volume of 20 µl of SCL at 23 °C for 15 min. NaOCl treatment has been shown to remove organic matter (41).
After treatments, the reaction mixtures were diluted to 1.0 ml with SCL
and 160-µl aliquots were removed and used for mineral formation
assays.
The fractionation scheme for the sequential
treatment of the CRMV to remove nonessential materials and reveal the
functionally active NC is outlined in Fig. 1.
Hyaluronidase and water treatments were used to remove
proteoglycan-related link protein and hyaluronic acid-binding protein.
Highly purified collagenase and 1 M NaCl in SCL were then
used to remove residual collagen and its fragments. To remove further
nonessential components, the vesicle remnants from the preceding steps
were then incubated with detergent, either CHAPS or Triton X-100, to
yield the NC.
Fig.
2 shows the time course of mineral formation by the NC
after CHAPS treatment. It is evident that mineralization of the NC was
actually increased compared to the untreated CRMV. Lowry analysis
indicated that only about one-third of the original protein remained in
the NC after the CHAPS treatment. However, chemical analyses revealed
that the Ca and Pi content of the MV was relatively unchanged; ~98% of the Ca2+ and ~86% of the
Pi were recovered in the NC.
Fig. 3 shows the SDS-PAGE
pattern of MV proteins in fractions from the sequential treatments
ending with CHAPS detergent, as depicted in Fig. 1. The protein profile
of the NC (Pellet F) was much simpler than that of the original MV.
Annexin V (~33 kDa) was most abundant, but significant amounts of
unidentified high (>300 kDa), and low (~10 kDa) molecular mass
proteins, as well as several lesser bands: annexin VI (~66 kDa),
annexin II (~36 kDa), alkaline phosphatase (~80 kDa), and three
unidentified proteins of ~50, 45, and 38 kDa were seen. Because of
their tight association with the NC, it is possible that these proteins
may regulate (influence) the rate of growth, shape, and size of
crystals during the nucleational process.
Prior to detergent extraction (four-step
extracted CRMV), the residual pellet had a lipid profile similar to
that of the original CRMV when analyzed by HPTLC (Fig.
4A). A variety of phospholipids, phosphatidylethanolamine, phosphatidylinositol, PS,
lysophosphatidylethanolamine, phosphatidylcholine, and sphingomyelin,
as well as significant amounts of free cholesterol, fatty acids, and
triacylglycerols were present. However, upon extraction with detergent
(either CHAPS or Triton X-100), a large amount of lipid was removed.
The lipid profile of the NC after Triton X-100 extraction (Fig.
4B) contained significant amounts of free cholesterol and
fatty acids, and an unidentified nonpolar lipid migrating near to
monoacylglycerols, as well as small amounts of
phosphatidylethanolamine, PS, and phosphatidylcholine. A similar lipid
profile was seen in the NC after CHAPS extraction, but the amount of
residual phospholipid was further reduced.
To more rigorously remove organic constituents, CRMV were
treated with either hydrazine monohydrate (40), or with sodium hypochlorite (41); sodium hydroxide treatment was used as a non-oxidizing, highly alkaline control (Fig. 5). It is
important to note here that hydrazine treatment has been shown not to
damage either ACP or HAP (40); hypochlorite treatment has been shown not to damage HAP crystals (41). A marked reduction in Ca2+
uptake was noted after hydrazine treatment, whereas after treatment with NaOCl, nearly all Ca2+ uptake ability of the CRMV was
destroyed. In contrast, NaOH treatment had no effect (10 mM) or stimulated (100 mM) mineral formation. Chemical analysis of the hydrazine-treated vesicles revealed that essentially all of the Ca2+ and Pi were
retained, but about 22% of the total P was removed, presumably from loss of phospholipids and phosphoproteins. Hydrazine removed ~95% of the proteoglycan associated with the MV and ~85% of the Lowry protein. Elemental composition of the hydrazine core by
EDAX analysis revealed Ca and P in nearly equimolar levels (see
below).
X-ray diffraction was used to
determine the crystallinity of the NC and to follow the pattern of
mineral accumulation when the NC was incubated in SCL. At 0 h,
with both the Triton X-100 and the CHAPS NC, a broad, diffuse
diffraction spectrum unlike that of synthetic ACP was observed; it
appeared to be derived primarily from organic material present in the
NC (data not shown). After incubation in SCL for 1-3 h, small
diffraction bands appeared in the region of 32° and 25.8° 2
The NCs were examined by TEM to reveal their
ultrastructure. At 90,000 × magnification, the CHAPS-NC appeared
to be composed of clusters of particles in aggregates ranging in size
from ~10 to 110 nm in diameter (Fig. 7A).
At 450,000 × magnification, these aggregates appeared to be made
of branched chains of particles 6.5-7.0 nm in diameter
(arrows) in arm-like extensions from the main body of the
aggregate (Fig. 7B). At 900,000 × magnification, these
~7-nm particles appeared to be composed of clusters of individual subunits ~1.0 nm in diameter (Fig. 7C,
arrows).
When the Triton X-100 NC was examined at 90,000 × magnification,
aggregates ranging in diameter from ~10 to 100 nm were again seen
(Fig. 7D). At 360,000 × magnification, these
aggregates appeared to be composed of small hollow spheres (~7 nm
outer, ~3 nm inner diameter, arrows) (Fig. 7E).
At 600,000 × magnification, these hollow spheres appear to be
composed of subunits ~1.0 nm in diameter (Fig. 7F). Thus,
although there were differences in general appearance, the basic
structures of both the CHAPS and the Triton X-100 cores were similar.
The ~1.0-nm dot-like subunits appear to be the smallest functional
component of nucleational core of MV. Their size, and the overall
Ca2+ and Pi composition of the NC (see below),
indicate that they may be clusters of Ca2+ and
Pi (HPO42 Well defined clusters of the detergent-extracted NC were
also analyzed by EDAX to determine their elemental composition. For the
CHAPS-treated NC, the relative abundance in atomic percent of the major
elements detected was: Ca, 51.4 ± 0.1%; P, 48.6 ± 0.1%
(mean ± S.E.). This gave a Ca:P ratio of 1.058 ± 0.005. For the Triton X-100-treated NC, the atomic percentages were: Ca, 51.6 ± 0.1%; P, 48.4 ± 0.1%, giving a Ca:P ratio of 1.066 ± 0.004. EDAX analysis of the hydrazine core gave the following atomic percentages: Ca, 51.8 ± 0.5%; P, 48.2 ± 0.5%, giving a
Ca:P ratio of 1.076 ± 0.022. Thus, the NC obtained after
extraction with each detergent, or with hydrazine, yielded material
with very low Ca/P molar ratios close to that of brushite. However in
each case, electron diffraction showed the material to be amorphous (data not shown).
FT-IR analyses of the NC after CHAPS
extraction (Fig. 8A) revealed a strong P-O
stretch band at 1054 cm
When the material remaining after treatment of CRMV with hydrazine was
examined by FT-IR, a pattern with greatly attenuated organic
constituents was seen. There was a broad O-H stretch band at 3400 cm The observation of a peak at 1238 cm To directly verify the presence of CPLX in the NC, without stripping
the MV with detergents, solid-state 31P NMR analyses of
lyophilized CRMV were performed (Fig. 9). Bloch decay
spectra of the MV (Fig. 9A) had a somewhat asymmetric
central peak at 3.2 ppm, surrounded by an asymmetric pattern of six
spinning sidebands of varying intensity. The central peak of this Bloch decay spectra is characteristic of
PO43
In this study, we focused on identification and characterization
of the NC present in MV. Previous FT-IR and FT-Raman spectroscopic studies had failed to detect the presence of well defined P-O stretch
or bending bands in freshly isolated MV (13, 14), even though the
vesicles were rich in Ca2+ and Pi (7).
Apparently, the Pi initially present in MV was masked by
large amounts of organic constituents. Therefore, in this paper we used
a sequence of extraction steps, digestions with purified enzymes, and
treatments with detergents, to remove interfering organic material from
the MV. The residual mineral-rich NC was then examined by a variety of
physicochemical methods to characterize its structure and
composition.
The majority of the original Ca2+ and Pi were
recovered in the NC, but only about 30% of the original protein.
SDS-PAGE analyses of the NC revealed that annexin V (33 kDa) was the
principal protein, although several others, including annexins II and
VI (36 and 67 kDa) and alkaline phosphatase (~80 kDa), were also
present (Fig. 3, and Ref. 15). Distinct bands at 45, 50, and >300 kDa were also seen (Fig. 3). Recent studies indicate that a 50-kDa protein
binds to annexin V in the presence of The role of the annexins present in the NC will now be considered. By
virtue of their well known acidic phospholipid-dependent Ca2+-binding properties (47), it is not surprising that
they were intimately associated with the mineral-rich NC (15). In fact, the majority of the annexins in MV can only be released by extraction with EGTA (48); however, some can be removed by extraction with acidic
chloroform-methanol, which also removes mineral-associated acidic
phospholipids (49). Thus in MV, annexins are tightly associated with
the mineral and the acidic phospholipid, PS, and exhibit
proteolipid-like properties. Recent studies indicate that annexin V may
be important for the orientation of Ca2+ and Pi
in the NC, stimulating crystal growth (33).
While the sequential enzyme and salt treatments removed little of the
original lipid, the detergents extracted most of the polar lipid from
the MV. In view of their amphipathic properties, it seems probable that
these detergents displaced and/or substituted for the polar lipid,
stabilizing the NC. However, they left behind much of the free and
esterified cholesterol, free fatty acid, and an unidentified lipid
migrating near monoacylglycerol, and critical small amounts of PS (Fig.
4). The persistence of sizable amounts of nonpolar lipids in the NC
indicates that they are difficult to extract with detergents. That they
may have a function is indicated by recent studies by Sarig et
al. (50), who suggest that cholesterol plays a role in
pathological mineralization.
Previous reports (23-27) had noted that the acidic phospholipids
interact and form complexes with the initial mineral phase, yet data
presented here indicate that these lipids can be removed by detergent
extraction. Since the NC was shown to be rich in calcium and phosphate,
we assessed its nature by direct analysis of various native MV
preparations (i.e. no treatment after isolation), using
solid-state 31P NMR. We compared these spectra with those
of synthetic Ca2+-PS-Pi complexes and calcium
phosphate mineral standards (38). These studies (Fig. 8) provided
important insight into the nature of the P-rich NC. All 0-h MV samples
had spectra similar to those of mixtures of ACP and synthetic CPLX,
when examined by proton cross-polarized 31P NMR. The
distinct central peak at 3.2 ppm is characteristic of ACP or HAP, but
diffraction studies showed no evidence of crystallinity. The proton
cross-polarization spinning sideband pattern of the CRMV was highly
distinctive and matched that of the CPLX; it did not resemble that of
any of the known crystalline calcium phosphates (45). By measurement of
the relative intensities of the central peak, and the spinning
sidebands of the CRMV and known mixtures of ACP and CPLX, the relative
abundance of the two phases could be assessed. These estimates gave a
value of 8.2% of the total vesicle P as CPLX and 91.8% as ACP. This
proportion of the total P as CPLX is in very close agreement with the
7% value obtained from solvent partition of MV by Wuthier and Gore
(26).
FT-IR, x-ray and electron diffraction analyses, and TEM of the NC all
revealed that the initial mineral phase was noncrystalline. The broad
unsplit P-O stretch pattern of the FT-IR spectra of both the CHAPS and
Triton-NC at 560 cm The CHAPS-NC had branched chains of aggregates in structures resembling
the high molecular weight sulfated proteoglycans (52). The formation of
these aggregates may be related to the high molecular weight band seen
in SDS-PAGE analysis of the CHAPS core (Fig. 3), but EDAX analyses did
not show any S in the NC. TEM of the Triton X-100 NC revealed the
presence of numerous hollow spheres ~7 nm in outside diameter. These
are similar in structure and size to those seen with synthetic
PS-Ca2+-Pi complexes (53) or with ACP prepared
by precipitation from aqueous media (54). At higher magnification, the
walls of these hollow spheres appeared to contain the ~1.0-nm subunit
particles. We interpret these findings to indicate that the hollow
spheres are composed of a residual lipid (and/or detergent) in which
are absorbed the ~1.0-nm clusters of Ca2+ and
HPO42 The marked enhancement in the 31P NMR spectrum by proton
cross-polarization, and the coupling constants observed (38), can only
be accounted for by the presence of
HPO42 FT-IR spectra of the synthetic CPLX revealed the presence of P-O
stretch peaks characteristic of crystalline calcium phosphates, but it
was not crystalline to electron diffraction. EDAX analyses of pure CPLX
reveal a Ca/P stoichiometry of 1:2 (i.e. Ca2+:1
PS:1 Pi). Taken together these data suggest that the NC
contains CPLX with Ca2+ bound to the phosphodiester group
of PS with bond orientations and energies that mimic the short range
order of crystalline calcium phosphates. However, diffraction data
indicate a lack of long range order seen in the crystalline state.
Recent solid-state 31P NMR studies of bone mineral (56)
have revealed the presence of a unique type of protonated phosphate (HPO42 To conclude, major advancement has been made in the structural and
physicochemical characterization of the NC of MV. It is now clear that
the initial mineral phase is primarily composed of a complex of the
acidic phospholipid, PS, and a noncrystalline calcium acid
phosphate. However, adsorbed to this complex are additional lipids such
as free cholesterol, the lipid-dependent Ca2+-binding proteins (the annexins), and some other
proteins still to be characterized. Given the complexity of this core
material, much additional work will be required to fully characterize
its remaining constituents and how they contribute to NC
properties.
We express appreciation to Theresa Litchfield
for assistance in preparing and analyzing some of the samples for
FT-IR.
Laboratory for Biomineralization Research,
Battelle Pacific Northwest Laboratories, Environmental & Molecular Science Laboratory, Richland, Washington 99352
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
-rich mixture of amorphous
calcium phosphate and a complex of PS, Ca2+, and
Pi. Taken together, our findings indicate that the NC of MV
is composed of an acid-phosphate-rich amorphous calcium phosphate intermixed with PS-Ca2+-Pi, annexin V, and
other proteins and lipids.
Isolation of Matrix Vesicles
, 63.5 mM sucrose, 16.5 mM TES, 12.70 mM K+, 5.55 mM D-glucose, 1.83 mM
HCO3
, 0.57 mM
Mg2+, and 0.57 mM
SO42
. The trypsin solution was
removed; tissue slices were rinsed twice with SCL, and then digested
with collagenase (200 units/g of tissue, type IA, Sigma) at 37 °C
for 3-3.5 h. The partially digested tissue was vortexed, and the
suspension was differentially centrifuged at 13,000 × g for 20 min and at 100,000 × g for 1 h. The MV pellet was resuspended as a stock suspension of 5.0 mg of
vesicle protein/ml of SCL containing 1.0 mM
Ca2+ (one-half the normal level of Ca2+ in SCL)
to prevent dissolution of labile Ca2+ and Pi
present (9), and also to minimize Ca2+ uptake by the
vesicles during storage. Protein levels in the vesicles were determined
by the method of Lowry et al. (29).
Fig. 1.
Sequential extraction scheme for isolating
the nucleational core from MV.
[View Larger Version of this Image (33K GIF file)]
1 using a Perkin Elmer model 1600 series spectrometer (13). (Some samples were rapidly frozen in liquid
N2 and not dried; they were examined at
196 °C by
solid-state NMR.) Solid-state 31P NMR spectra of the CRMV
and the synthetic PS-Ca2+-Pi complex (33) were
obtained using a Varian Unity+ spectrometer operating at 121.42 mHz for
31P. Pulse widths were 4.4 µs, and a contact time of 2.5 ms was used. Samples (10-20 mg) were packed into 7-mm Doty Scientific SiN3 rotors and spun at 2 kHz, stabilized to a precision of
±1 Hz. Chemical shifts were referenced to external
H3PO4 at 0 ppm (38).
radiation, a nickel filter, 40 kV and 20 mA, 1° receiving slit, and a scanning speed of 1/8° 2
/min
(39).
Fractionation
Fig. 2.
Mineral phase induction by CRMV and the
CHAPS-nucleational core. CRMV were sequentially extracted as shown
in Fig. 1. Samples of the original CRMV and the final CHAPS-NC were
incubated in SCL, and their ability to induce mineral formation was
measured by light scattering at 340 nm as described under
"Experimental Procedures." Note that the NC actually formed more
mineral than did the original CRMV.
[View Larger Version of this Image (23K GIF file)]
Fig. 3.
SDS-PAGE analysis of the proteins present in
CRMV and in the various sequential subfractions. CHAPS (1% in
SCL) was used as the detergent. The letters on the gel
correspond to those shown in Fig. 1. Pellet A, original
CRMV; Pellet B, after digestion with hyaluronidase (not
shown); Pellet C, after extraction with deionized distilled
water; Pellet D, after digestion with collagenase; Pellet E, after extraction with 1 M NaCl in SCL;
Pellet F, after extraction with CHAPS. Supernatant
A, after digestion with hyaluronidase; Supernatant B,
after extraction with deionized distilled water; Supernatant
C, after digestion with collagenase; Supernatant D, after extraction with 1 M NaCl in SCL; Supernatant
E, after extraction with CHAPS. Note the selective removal of
proteins (seen in the supernatants) by the sequential extraction steps.
Note in Pellet F the much cleaner and simpler protein
pattern of the nucleational core compared to the CRMV. Note that the
33-kDa band (annexin V) is the dominant protein in the NC.
[View Larger Version of this Image (86K GIF file)]
Fig. 4.
HPTLC analysis of the lipids present in CRMV
and in the various sequential subfractions. A, the lipid
composition of the original CRMV pellet and that of the pellet after
the first four treatments prior to detergent extraction.
B, the lipid composition of the original CRMV pellet and
that of the pellet after the specified detergent extraction.
The various CRMV fractions were first decalcified by treatment with
EDTA and then extracted with organic solvents as described under
"Experimental Procedures." Extract 1 denotes lipids that
were extracted with chloroform-methanol (2:1, v/v); Extract
2 denotes lipids extracted subsequently with
chloroform-methanol-concentrated hydrochloric acid, (200:100:1, v/v/v).
Panel A, lane 1, standards I, in descending
order: free fatty acids (FFA), glucosyl ceramide (GC), phosphatidylinositol (PI), and
sphingomyelin (SM); lane 2, standards II, in
descending order: cholesterol esters (CHE), triacylglycerols
(TG), free cholesterol (CH) (and overlapping, 1,2-diacylglycerols (DG)), monoacylglycerols
(MG), phosphatidylethanolamine (PE),
phosphatidylserine (PS), and phosphatidylcholine
(PC); lane 3, standards III,
lysophosphatidylserine and other lysophospholipids (LPS, not
clear in this TLC); lane 4, CRMV, Extract 1; lane
5, CRMV, Extract 2; lane 6, four-step treated CRMV,
Extract 1; lane 7, four-step treated CRMV, Extract 2. Panel B, lane 1, standards I, in descending order:
cholesterol esters (CHE), free fatty acids (FA),
free cholesterol (CH), glucosyl ceramide (GC),
phosphatidylinositol (PI), sphingomyelin (SM);
lane 2, Standards II, in descending order: triacylglycerols
(TG), 1,2-diacylglycerols (DG), monoacylglycerols (MG), phosphatidylethanolamine (PE),
phosphatidylserine (PS), phosphatidylcholine
(PC); lane 3, Standards III, in descending order:
lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS),
lysophosphatidylcholine (LPC); lane 4, CRMV,
Extract 1; lane 5, CRMV, Extract 2; lane 6,
CHAPS-NC of CRMV, Extract 1; lane 7, CHAPS-NC of CRMV,
Extract 2; lane 8, Triton-NC of CRMV, Extract 1; lane
9, Triton-NC of CRMV, Extract 2.
[View Larger Version of this Image (42K GIF file)]
Fig. 5.
Effect of hydrazine, sodium hypochlorite, and
sodium hydroxide treatment on mineral formation by CRMV. CRMV were
treated with hydrazine monohydrate, sodium hypochlorite, or sodium
hydroxide to dissolve organic matter, as described under
"Experimental Procedures," and incubated in SCL for measuring
mineral formation. Note after hydrazine treatment, that while mineral
formation occurred more quickly than normal after introduction into the
SCL, the period of rapid mineral formation was less extended and at a
slower rate than that of native CMRV. After 100 mM sodium
hypochlorite treatment (pH 12), there was no appreciable mineral
formation for the duration of the assay, whereas 10 mM
NaOH, (pH 12) had little effect on the vesicle's activity. Also note
that 100 mM NaOH (pH 13) stimulated both the rate and
extent of MV-mediated mineralization. This indicates that there was no
crystalline mineral present in the CRMV, otherwise mineral formation
would have occurred rapidly and extensively.
[View Larger Version of this Image (25K GIF file)]
indicative of the formation of poorly crystalline apatite. After
16 h incubation, there were increased amounts of diffractive
material (Fig. 6). The mineral phase may be a poorly
crystalline carbonate hydroxyapatite, similar to that seen in other
biological minerals (18, 20).
Fig. 6.
X-ray diffraction analysis of the mineral
formed by the nucleational core after incubation in SCL. CRMV were
sequentially treated as shown in Fig. 1, and then extracted with either
CHAPS or Triton X-100 to yield the residual cores, which were incubated in SCL. At the specified times, the incubation mixture was centrifuged at 100,000 × g for 1 h and the pellet lyophilized
and analyzed by micro x-ray diffraction. A, CHAPS-NC
incubated for 1 h in SCL; B, CHAPS-NC incubated for
16 h; C, Triton X-100-NC incubated for 1 h in SCL;
D, Triton X-100-NC incubated for 16 h. Note the buildup of the diffracting mineral phase after the 16-h incubation, although there is already detectable crystalline mineral present after 1 h
of incubation. Before incubation with SCL, the diffraction patterns of
the NC were very broad and no peaks characteristic of crystalline
phosphates were evident (data not shown).
[View Larger Version of this Image (23K GIF file)]
Fig. 7.
TEM analysis of the nucleational core
obtained by sequential extractions and finally detergent
treatment. CRMV were sequentially treated as described in Fig. 1
to yield the CHAPS-NC (A-C) or the Triton-NC
(D-F). After lyophilization, small samples were dispersed
in 100% ethanol, and one drop of the alcoholic suspension was mixed
with one drop of SCL and immediately transferred to carbon-coated grids
and dried. Grids were not stained. The structures of the NC were
examined by TEM at successively higher magnifications (see
"Experimental Procedures"). CHAPS-NC: A, 90,000 × (bar = 200 nm); B, 450,000 × (bar = 40 nm);
C, 900,000 × (bar = 20 nm). Triton-NC:
D, 90,000 × (bar = 200 nm); E,
360,000 × (bar = 50 nm); F, 600,000 × (bar = 30 nm). In A, the arrow points to an
aggregate of particles ~110 nm in diameter. In B, the
arrows point to particles ~6 nm in diameter at the end of
chains. In C, the arrows point to areas where
clusters of individual subunits ~1.0 nm in diameter can be seen to
make up the particles. (Some isolated subunits can be seen between some
of the clusters of particles in B). In D, the
arrow points to an aggregate of particles ~100 nm in
diameter, which in E can be seen to be hollow spheres (arrows) ~7 nm outer, ~3 nm inner diameter. In
F, careful inspection reveals that walls of the hollow
spheres are composed of subunits (arrows) ~1.0 nm in
diameter.
[View Larger Version of this Image (148K GIF file)]
) ions stabilized
by lipids (detergent) and proteins.
1 with shoulders at 1096 and 1000 cm
1 and a broad unsplit P-O absorption band (558 cm
1) characteristic of non-crystalline calcium phosphate
minerals. There were the strong O-H stretch bands at 3332-3507
cm
1 of hydrates, strong C-H stretch bands of lipids at
2921.5 and 2851.7 cm
1, as well as the strong amide bands
of proteins (1654 and 1544 cm
1). The NC after Triton
X-100 extraction (Fig. 8B) had an FT-IR spectrum similar to
that seen with CHAPS. In both NCs there was a well defined peak at 1238 cm
1 characteristic of Ca2+ bound to the
O-P-O phosphodiester of the polar head group of PS (43).
Fig. 8.
FT-IR analysis of the nucleational core and
of a synthetic PS-Ca2+-Pi complex. CRMV
were sequentially extracted to yield the CHAPS-NC or the Triton
X-100-NC as described in Fig. 1. The PS-Ca2+-Pi
complex was synthesized as described previously (33). All samples were
lyophilized, and ~1 mg of each was incorporated into ~300 mg of
KBr, compressed into a transparent pellet, and examined by FT-IR.
A, CHAPS-NC; B, Triton X-100-NC; C,
synthetic CPLX. The band at ~1235 cm1 seen in the CHAPS
and Triton X-100 NCs and the CPLX is characteristic of
Ca2+-PS complexes (43).
[View Larger Version of this Image (25K GIF file)]
1; however, the C-H stretch region of the lipid acyl
chains and the two amide bands of proteins were much reduced. The P-O
stretch bands of mineral phase phosphates were strong (data not shown). NaOCl-treated MV were not examined by FT-IR because previous studies by
Tomazic et al. (44) had shown that hypochlorite treatment altered all mineral phases except HAP.
1 (Fig. 8,
A and B) suggested the presence of
Ca2+ complexed with PS in both the CHAPS and Triton X-100
NCs, a finding consistent with earlier data indicating the presence of
CPLXs in MV (26). Thus, FT-IR spectra of synthetic CPLXs were also obtained. All samples of CPLX had a distinct peak at ~1238
cm
1 (Fig. 8C). Other features were: strong
C-H stretch bands at 2852 and 2922 cm
1 typical of lipid
acyl chains, strong peaks at 1740, 1637, and 1618 cm
1
from the C=O groups of acyl esters and free carboxylate groups of PS,
strong P-O stretch peaks at 1035-1101 cm
1, and two
sharp peaks in the P-O absorption region at 561 and 600 cm
1. CPLX isolated after brief (<10 min) reaction of
Ca2+ with PS and Pi, showed only a broad band
in the 1035-1101 cm
1 region.
groups (e.g. in ACP),
but the six asymmetric sidebands indicated that this was not the only
solid phase. Proton cross-polarization 31P NMR spectra of
the MV (Fig. 9B) greatly enhanced the intensity of the
sidebands, indicating an abundance of acid phosphate (i.e. HPO42
) groups in the solid phase. The
complex pattern of spinning sidebands seen in MV was unlike that of any
known crystalline calcium phosphate (45); however, it was similar to
that observed for synthetic CPLX (Fig. 9C).
Fig. 9.
Solid-state 31P NMR analysis of
native CRMV. Lyophilized CRMV (10-20 mg) were packed into 7-mm
Doty Scientific SiN3 rotors, spun, and recorded at room
temperature as described under "Experimental Procedures." Bloch
decay and cross-polarization pulse sequences were employed with high
proton decoupling; 10-s recycle time was used for the Bloch decay, and
3-s times were used for the cross-polarization experiments.
A, CRMV, Bloch decay; B, CRMV, proton
cross-polarization; C, CPLX, proton cross-polarization. Note
the marked increase in intensity of the spinning sidebands in
B compared to A, when subjected to proton
cross-polarization. Note the close similarity in the position and
relative intensity of the spinning sidebands of the CPLX and CRMV upon
proton cross-polarization. The large central band in A and
B is due to the presence of ACP. Based on the relative intensity of the central peak and the sidebands, compared to known mixtures, ~8% of the P in the CRMV is composed of the CPLX.
[View Larger Version of this Image (20K GIF file)]
800 nM
Ca2+, preventing its removal from membranes (46). In view
of the abundance of annexin V and Ca2+, the 50-kDa protein
in the NC may be related to this protein. The >300-kDa protein seen in
the SDS gels of the NC remains to be identified.
1 is characteristic of noncrystalline
calcium phosphates. X-ray diffraction and electron diffraction analyses
similarly failed to detect significant crystallinity. TEM examination
revealed that both the CHAPS- and the Triton X-100 cores were composed of small 6-8-nm diameter aggregates composed of small dot-like subunits ~1.0 nm in diameter, similar to those previously described by Höhling et al. (51).
/PO43
ions. The ~1.0-nm diameter of the dot-like subunits is similar in
size to the core structure of ACP measured by radial distribution function analysis (42, 55).
, not by adjacent H2O
molecules. These findings reveal that the NC of native MV must contain
substantial levels of protonated (HPO42
) phosphate. This is in
agreement with EDAX analyses of the NC. Ca2+/Pi
ratios from ten separate areas of the CHAPS and Triton X-100 NC were
1.062 ± 0.010 (mean ± S.D.); those of the hydrazine core from which the most organic phosphate was removed were 1.076 ± 0.039. Since EDAX showed no significant other elements (e.g.
no sodium, magnesium, or potassium), these values indicate that the NC
contains calcium phosphates with predominantly
HPO42
(see NMR data). Assuming that
ACP was composed of mixtures of both
Ca9(PO4)6 (deprotonated phosphate,
Ca/P ratio = 1.50) and CaHPO4 (Ca/P = 1.00) the
relative abundance of these two forms can be estimated. Taking into
account the presence of the CPLX (~8% of the P), the
HPO42
form appears to represent about
71% of the total P in MV.
) group, which has an
isotropic chemical shift precisely like that of OCP, and not
like that of brushite. In contrast, its anisotropic chemical
shift is similar to that of brushite, but significantly different from
that of OCP. It is possible that this protonated phosphate may be
related to the CPLX complex we have identified in MV. This idea is
supported by our previous finding that the early mineral induced by MV
has characteristics, when examined by FT-IR and FT-Raman, like that of
OCP (13, 14).
*
This work was supported by Grant AR18983 from the NIAMS,
National Institutes of Health. 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 Chemistry and
Biochemistry, 424A Physical Sciences Center, Columbia, SC 29208. Tel.:
803-777-6626; Fax: 803-777-9521; E-mail: wuthier{at}psc.sc.edu.
1
The abbreviations used are: MV, matrix
vesicle(s); NC, nucleational core; FT-IR, Fourier transform-infrared;
FT-Raman, Fourier transform-Raman; ACP, amorphous calcium phosphate;
SCL, synthetic cartilage lymph; OCP, octacalcium phosphate; HAP,
hydroxyapatite; PS, phosphatidylserine; CPLX,
Ca2+-PS-Pi complex; TEM, transmission electron
microscopy; EDAX, energy-dispersive analysis with x-rays; HPTLC, high
performance thin-layer chromatography; PAGE, polyacrylamide gel
electrophoresis; CRMV, collagenase-released matrix vesicle(s); TES,
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.