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J. Biol. Chem., Vol. 278, Issue 34, 32047-32057, August 22, 2003
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From the
Departments of Biochemistry and
Biophysics, ||Orthopaedic Surgery, and
**Cell and Developmental Biology, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the
¶Department of Pathology and Laboratory Medicine,
Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903
Received for publication, May 2, 2003 , and in revised form, May 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10–6% of the dry weight of
tissue, making it by far the least abundant collagen ever isolated.
Transmission electron microscopy after rotary shadowing revealed the
appearance of rodlike structures with multiple sharp bends, a small nodule at
one end of the molecule, and a total length of 240 nm. Domain-specific
antibodies were used to identify the nodule as the noncollagenous amino
terminus, whereas the location of most kinks corresponds to major
interruptions separating the five collagenous subdomains. More than half of
the type XIX molecules observed were present in oligomers of different size
and complexity, resulting from association of the amino-terminal domains.
Biochemical analysis demonstrated that these supramolecular aggregates are
dependent upon and/or stabilized by intermolecular disulfide cross-links and
that the globular amino terminus contains a high affinity, heparin-binding
site. The polymorphic conformational states of this rare collagen, and its
ability to self-assemble into a higher order structure provide focal points
for future determination of biologically significant functions in cell-cell
and/or cell-matrix interactions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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333 continuous
Gly-X-Y triplets and involved in the formation of
cross-striated fibrils) and the nonfibrillar group, a highly heterogeneous
class exhibiting a spectrum of sizes, supramolecular assemblies, and chain
organization, with the one commonality being the presence of noncollagenous
sequences interrupting and/or flanking collagenous domains
(1,
2,
8–10).
Understanding the complex structure and function of these many proteins has
proven to be a formidable task despite, in many instances, extensive knowledge
of disease phenotypes directly attributable to the respective collagen gene
mutations (1,
11,
12). A major complication in
this process has been the scarcity of a number of collagen types and the
inability to directly characterize the in vivo form of the molecules.
One of these elusive and increasingly intriguing collagens is type XIX.
Type XIX collagen was identified from independently isolated clones
representing RNA purified from a human rhabdomyosarcoma
(RMS)1 cell line
(13,
14). The type XIX chain is
composed of a 268-residue, noncollagenous amino terminus, an 832-residue
discontinuous collagenous region, and a 19-residue carboxyl peptide
(14–16).
Several features in the type XIX sequence place this collagen in the largest
subclass of the nonfibrillar group, together with types IX, XII, XIV, XVI, XX,
and XXI
(2–4,
10). These include an
250-residue thrombospondin module in the amino terminus (Tsp-N), the
position of two 2-amino acid interruptions in the collagenous subdomain
closest to the carboxyl terminus, and a Cys-Xaa4-Cys motif situated
at the junction of the collagenous region and carboxyl peptide
(10,
15,
16). By virtue of four
internal 20–44-residue interruptions, the primary sequence of the unique
type XIX collagenous region can be divided into five 70–224-residue
subdomains; four include a few short interruptions, and the fifth is composed
solely of Gly-X-Y triplets
(14–16)
(illustrated in Fig.
12A). In contrast, only two or three collagenous
subdomains are found in other members of the above mentioned subclass, except
for type XVI, which is interspersed with a heterogeneous array of
9–10 generally less well defined collagenous segments
(2–4,
9,
10,
17).
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To begin direct exploration of this vital protein at the ultrastructural
level, we have purified the native tissue form of type XIX and characterized
the molecule by electron microscopy and biochemical analysis. Type XIX
collagen in human umbilical cord is extremely scarce (i.e.
10–6% of dry weight, which is several orders
of magnitude less than any other collagen so far isolated). Electron
microscope images revealed a sharply kinked and highly polymorphic collagenous
region and the existence of higher order complexes. The involved
amino-terminal domain is responsible for intermolecular disulfide linkages and
contains a proven heparin-binding site.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of Type XIX Collagen—Human umbilical cords were
obtained from the Hospital of the University of Pennsylvania. About 400 cord
specimens totaling 8 kg and weighing 3.5–56 g each were collected
for this project. Cords were frozen temporarily at –20 °C and
transferred to –80 °C. For large scale preparations, 10–15
specimens (250 g, wet weight) were pooled and processed as described
below; for rotary shadowing, two preparations were combined before the
antibody affinity column. Processed tissue, protein samples, and buffers were
maintained in an ice slurry when possible or else kept at 4 °C. Tissue was
thawed on ice, washed several times in buffer (50 mM Tris-HCl, 4.5
M NaCl, 20 mM EDTA, pH 7.5, 10 mM
N-ethylmaleimide, and 0.5 mM phenylmethylsulfonyl
fluoride), cut into 0.5-cm pieces, and added to extraction buffer (10:1 (v/w)
50 mM Tris-HCl, 1.0 M NaCl, 10 mM EDTA, pH
7.5) with protease inhibitors (10 mM N-ethylmaleimide, 0.5
mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml
aprotinin, all obtained from Sigma). The tissue was homogenized at speed 5
(speed 10 = 27,000 rpm) for 8 x 1 min (Polytron; Brinkman Instruments,
Westbury, NY). The tissue suspension was stirred slowly for 21–24 h and
centrifuged at 32,000 x g for 30 min. Supernatant proteins were
precipitated overnight with the addition of ammonium sulfate to 40%
saturation, centrifuged as above, resuspended in 200 ml of extraction buffer
containing 0.1% Triton X-100, stirred overnight, and dialyzed for 4 h against
2 liters of buffer containing 25 mM Tris-HCl, 0.4 M
NaCl, 2.0 mM EDTA, pH 7.4, 0.1% Triton X-100, and the inhibitors
listed above. The sample was then dialyzed against the same buffer except with
a lower pH (7.2) and NaCl (0.1 M) concentration. The mixture was
clarified at 20,000 x g for 15 min, and the supernatant was
incubated in a batch procedure with 40 ml of SP Sepharose Fast Flow resin
(Amersham Biosciences) pre-equilibrated in the final dialysis buffer
containing 0.1 M NaCl. The protein solution and resin were
incubated on a rocker overnight and centrifuged at 480 x g for
5 min, and the resin was washed/eluted stepwise at 30-min intervals using 3
x 140 ml of the binding buffer and 4 x 80, 4 x 40, and 2
x 40 ml of the same buffer containing 0.3, 0.6, and 1.0 M
NaCl, respectively. Type XIX collagen in the first two portions of the 0.6
M NaCl eluate was concentrated (16–20-fold) (Ultracell
Amicon YM 30 Ultrafiltration Discs; Millipore Corp.) and then brought to a
final concentration of 0.4 M NaCl. The BCA reagent (Pierce) was
used for protein assays.
The type XIX pool was mixed with Affi-Gel 10 resin (4:1, v/v) to which the purified COOH-Ab was bound. The resin was pre-equilibrated in 0.4 M NaCl buffer containing 25 mM Tris-HCl, 2.0 mM EDTA, pH 7.2, 10 mM N-ethylmaleimide, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin (or 1 µg/ml pepstatin A), and 0.1% Triton X-100. The sample was incubated with the resin on a rocker platform for 8–16 h. The column was washed with 4 x 3mlof0.4 M NaCl binding buffer, and type XIX collagen was eluted stepwise in 0.8–1-ml fractions of 1.0 M glycine, pH 2.7, immediately neutralized with 1 M Tris base. Fractions were collected by gravity in polypropylene tubes precoated with Triton X-100 (0.1%). Before elution, Triton X-100 and EDTA, pH 7.2, were added to each tube in order to adjust the final concentration in each fraction to 0.1% and 2.0 mM, respectively. Protease inhibitors were added to each fraction immediately after collection. Fractions were aliquoted, frozen in dry ice, and stored at –80 °C, or the peak fraction was dialyzed against 0.1 M ammonium formate, pH 7.4, 1.0 mM EDTA, 0.1% Triton X-100, and protease inhibitors for 16 h for rotary shadowing. The microdialyzer (Spectrum, Gardena, CA) and membrane (molecular mass cut-off of 50 kDa) were pretreated with 0.1% Triton X-100.
Bacterial Collagenase Digestion—Collagenase digestion was carried out for 75 min at 37–38 °C in a 15-µl reaction containing 50 mM Tris-HCl, pH 7.2, 10 mM calcium acetate, and 2–5 units of bacterial collagenase form III (Advance Biofactures, Lynbrook, NY). Control samples (undigested) were incubated in the same buffer without collagenase.
Gel Electrophoresis and Western Blot Analysis—Samples were boiled for 2 min in 60 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 50 mM EDTA, 0.025% bromphenol blue, 200 mM DTT and electrophoresed in 6, 7, 10, or 12% polyacrylamide-SDS gels. Proteins were transferred to Immobilon-P membranes (Millipore) (18, 23). Membranes were incubated for 90 min with the primary antibody (0.1–1 µg/ml of the COOH-Ab or a 1:100–200 dilution of serum-free medium from the clone producing the monoclonal NH2-Ab), washed, and incubated with secondary antibodies (anti-rabbit IgG, peroxidase-linked F(ab')2 fragment from donkey or anti-mouse IgG, peroxidase-linked whole antibody from sheep). Membranes were developed using ECL reagents (Amersham Biosciences).
Silver Staining—Type XIX protein was stained using the SilverXpress silver staining kit from Invitrogen (Carlsbad, CA) following the company's procedure for "samples reduced with DTT" with the following modifications. Samples were brought to a final concentration of 20 mM DTT, boiled for 2 min, and cooled to room temperature. Iodoacetamide was added to 0.1 M, and samples were incubated at 37 °C for 10 min before being applied to the gel. In addition, during the gel staining process, the second sensitizing step was increased from 30 to 60 min.
Immunohistochemistry—Human umbilical cord was obtained from Robert Wood Johnson University Hospital, snap-frozen in OCT compound in methylbutane at liquid nitrogen temperature, and sliced into 4-µm sections. The immunoperoxidase staining procedure has been detailed earlier (18, 19). Sections were incubated with the type XIX COOH-Ab (18) or a type IV collagen polyclonal antibody (DAKOpatts, Carpentaria, CA). Swine anti-rabbit secondary antibody and peroxidase-conjugated streptavidin were also obtained from DAKOpatts.
Heparin-Sepharose Chromatography—An aliquot (220 µg) of the SP Sepharose pool was diluted to 0.2 M NaCl and digested to completion with 100 units of bacterial collagenase for 3 h at 37 °C in 0.5 ml. EDTA was added to a final concentration of 20 mM, and the sample was diluted to 1 ml and applied to a 1-ml heparin-Sepharose HiTrap column (Amersham Biosciences) in a Tris-HCl, pH 7.4, buffer containing 0.1 M NaCl, 10 mM EDTA, and protease inhibitors. The column was washed with 7 ml each of 0.1 and 0.3 M NaCl buffers. The bound material was eluted in 1 M NaCl buffer in 0.3-ml fractions.
Rotary Shadowing Electron Microscopy—Type XIX collagen was visualized by electron microscopy of rotary-shadowed samples prepared by modifications of published methods (24–26). Type XIX, eluted from the antibody affinity column, was dialyzed in ammonium formate as stated above, and autoclaved glycerol (99.5+% spectrophotometric grade; Aldrich) was added to give a final concentration of 50%. The sample was sprayed onto 0.5-cm2 pieces of freshly cleaved mica using an EFFA spray mount device (E. F. Fullam Inc., Latham, NY). The sheets of mica were then placed in a Denton DV-502 vacuum evaporator (Denton Vacuum, Cherry Hill, NJ) and pumped until the vacuum was about 3–4 x 10–7 torr. Tungsten was evaporated at an angle of about 4–7° while the stage containing the mica was rotating, and carbon was evaporated on top of the tungsten as a support layer. The replicas were floated off the mica onto a water surface and picked up onto 400-mesh copper grids and examined with a Philips EM 400T transmission electron microscope (Philips, Hillsboro, OR) at 80 kV. Many grids were examined, and micrographs were taken from a variety of areas at a magnification of generally x 60,000. In an alternative method (the Nagaswami technique) modified from previous reports (27), about 10 µl of the sample was applied to a piece of Parafilm on ice. A 0.5-cm2 piece of freshly cleaved mica was placed on the top of the droplet and allowed to remain on the ice for about 2 h. The mica was removed from the Parafilm, and the buffer described above (50% glycerol in ammonium formate buffer) was placed onto the mica. The excess solution was removed by blotting, and the mica was allowed to remain in the cold room overnight. The next day, rotary shadowing was carried out as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemical Staining of Type XIX in Umbilical Cord—To corroborate the Western blotting result and to identify what structures type XIX was associated with in cord tissue, immunohistochemical analysis was conducted. The umbilical cord is covered by a simple amniotic epithelium and contains one vein and two arteries surrounded by a mucous connective tissue matrix, Wharton's jelly (29–31). Type XIX was present in epithelial, smooth muscle, and endothelial BMZs (Fig. 2). The amniotic epithelial BMZ (upper left panel) and the BMZ surrounding the muscle cells of the vessels (upper right panel) were strongly reactive with the type XIX COOH-Ab. Within Wharton's jelly (upper left panel), type XIX staining showed a diffuse localization throughout the outer region, whereas in the portion of the cord adjacent to the large vessels, it appeared more condensed (data not shown). The BM/BMZ localization of type IV collagen (Fig. 2, lower panels) was comparable with type XIX, and in Wharton's jelly, the type IV staining was more uniform throughout the matrix (lower left panel).
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Purification of Type XIX Collagen by Neutral Salt Extraction and by Ion Exchange and Antibody Affinity Chromatography—A number of different sized umbilical cord pieces (usually 10–15) were pooled for each of the type XIX preparations; the maximum amount of tissue processed at one time was 250 g. Almost all of the type XIX collagen was extracted from homogenized tissue in a 1 M NaCl buffer, and subsequent concentration and fractionation using 40% saturated ammonium sulfate resulted in an excellent recovery (see "Materials and Methods"; data not shown). Following precipitation, the pellet was resuspended, dialyzed, and applied to an ion exchange resin. The choice was dictated by the 8.6 pI of the protein, and as predicted, type XIX exhibited a high affinity for the cation exchange resin, SP Sepharose (see "Materials and Methods"; data not shown). The protein was bound in a batch procedure at 0.1 M NaCl, completely retained after a 0.3 M NaCl wash, and eluted using 0.6 M NaCl, where >95% of the type XIX was recovered in the first two buffer applications. The ionic strength of the 0.6 M pools 1 and 2 corresponded to 0.44 and 0.55 M NaCl, respectively, reflecting mixture with the wash buffer. Western blotting of a final 1.0 M NaCl treatment of the resin was negative for type XIX.
In a silver-stained gel of the SP Sepharose 0.6 M NaCl pool, a probable type XIX band appeared as a very minor species in a heterogeneous array of various sized proteins (data not shown). This result verified the scarcity of type XIX and the absolute requirement for a specific mode of purification only afforded by an antibody affinity column. The SP Sepharose eluate was concentrated, diluted to 0.4 M NaCl, and mixed overnight with the type XIX COOH-Ab covalently bound to a gel matrix. The column elution profile was evaluated by Western blotting using both the NH2- and COOH-Abs (Fig. 3, A and B). A minimal amount of type XIX was found in the flow-through and wash (Fig. 3, lanes 2, 3, 9, and 10); type XIX eluted sharply in fraction 2, where the pH 2.7 glycine displaced the pH 7.2 equilibration and wash buffer (Fig. 3, lanes 5 and 12).
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Different type XIX cleavage fragments were detected, depending upon the
antibody used. Noticeable throughout the procedure, but somewhat increased in
the purified fraction, was a 120-kDa minor fragment identified by the
NH2-Ab (Fig.
3A, lanes 1 and 5). A much more
pronounced cleavage product was evident using the COOH-Ab; about one-third to
one-half (depending upon the preparation) of type XIX was present in the form
of a 140-kDa band that was seen before the column elution as a comparatively
minor fraction (Fig.
3B, lanes 8 and 12). Despite the
immediate addition of a variety of freshly prepared protease inhibitors to the
fractions, the 140-kDa species (later visualized by electron microscopy)
remained a significant portion of the type XIX recovered.
The yield and purity of type XIX was also determined by silver staining
(Fig. 4). The 165-kDa intact
chain and the 140-kDa degradation fragment were clearly seen; the 120-kDa
fragment was faintly evident (Fig.
4, lane 1). All bands were digested with bacterial
collagenase (Fig. 4, lane
2) and their identity and relative proportions verified by Western
blotting of aliquots electrophoresed on parallel lanes (data not shown). The
total amount of type XIX at this final step, too low for conventional protein
assays to be employed, was estimated by comparing the intensity of the bands
to a range of known amounts of type I collagen electrophoresed on adjacent
lanes (data not shown). The results showed that 1.5 µg of type XIX
protein was purified from 230 to 250 g of umbilical cord tissue, translating
to a representation of 6 x 10–6%. A flow
diagram of the procedure, together with absolute and relative amounts of
protein recovered at each step, is shown in
Fig. 5.
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The Unique Shape of Type XIX Collagen as Visualized by Electron Microscopy—The purified native type XIX collagen was examined by transmission electron microscopy following rotary shadowing with tungsten in a vacuum evaporator. Eight independent experiments were carried out using type XIX collagen alone and/or type XIX incubated with the COOH- or NH2-Ab. Visualization of the grids containing type XIX alone revealed that most of the individual particles were long, rodlike structures with multiple kinks or bends and a small nodule at one end (Fig. 6), indicating that this is the shape of the type XIX molecule. The dimensions of the structures observed were determined. The mean diameter of the globular region was 19.7 ± 2.6 nm (n = 40). The mean length of the rods could not be established directly because of the many different combinations and angles of the kinks. Instead, individual segments were measured, and a histogram was constructed to show the average positions of the kinks relative to the end of the molecule as defined by the globular region (Fig. 7). Six peaks (P1–P6) representing the sites of the major kinks were identified; they were located at 52 (P1), 70 (P2), 85 (P3), 135 (P4), 170 (P5), and 215 (P6) nm. Whereas these distances are the averages of all molecules containing each of these kinks, it should be noted that not all molecules contain all of the kinks (Fig. 6). Some peaks are broad, suggesting that there may be variability in the location of the kinks. Such differences can be accounted for if portions of some molecules were not lying flat on the surface when they were sprayed. In this case, the images observed would be projections of the individual rod segments with correspondingly shorter lengths, depending upon how steeply they were angled. Such behavior would be expected for rigid collagen rods. Some of the variation could also be explained because the bend could occur at different sites in the four large noncollagenous (NC) segments (20–44 residues) as well as within the nine smaller interruptions (1–6 residues) that occur in four of the five collagenous subdomains (a schematic diagram of the chain is shown in Fig. 12A). The mean total length of the whole molecule was estimated at 240 ± 23 nm.
Other structures commonly seen in the micrographs were long, thin, kinked rods without any globular domains at the end and globular nodules alone (data not shown). Many of the nodules were identical in size and shape to those at one end of the most commonly observed structures shown in Fig. 6. Other particles had the appearance of two or more nodules associating with each other. The rods were identical to the rod portion of the structures that also contained a nodule at one end (Fig. 6). It was apparent that the separated nodules and rods are, as described above in the purification process (Figs. 3 and 4), cleavage products of the larger, intact structures.
Polyclonal and monoclonal antibodies to the type XIX carboxyl- and amino-terminal ends, respectively, were used to identify those portions of the structures (Fig. 8). Rotary-shadowed IgG antibodies appear by electron microscopy to be three-lobed structures in which the relationship among the lobes is highly variable because of their flexibility (32). Many different views of various antibody conformations are visible. After incubation of the antibodies with type XIX collagen, the complexes were rotary-shadowed and studied by electron microscopy. The type XIX polyclonal COOH-Ab bound to the end of the collagen opposite from the nodule, verifying the location of the short carboxyl-terminal peptide (Fig. 8A). In contrast, images of type XIX collagen incubated with the monoclonal NH2-Ab show that this IgG molecule bound to the nodule itself, corroborating that this is the amino-terminal noncollagenous domain (Fig. 8B).
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Identification of Type XIX Amino-terminal Linked Complexes—In the electron micrographs of all preparations of type XIX, there were many aggregates of the collagen molecules. These almost invariably interacted with each other via their globular amino-terminal ends (Fig. 9). In most cases, the number of molecules in each aggregate could be extrapolated from the number of rodlike tails extending away from the core containing the interacting nodules. All molecules, both those interacting with each other and the individual ones, were counted in many micrographs, yielding an estimate of the proportion of multimers. Total percentages for each size oligomer (n = 300) were as follows: monomer, 43.7%; dimer, 25.9%; trimer, 8.9%; tetramer, 7.4%; pentamer, 7.4%; hexamer or larger, 6.7%. In some instances, these structures were too intricate to accurately assess the number of constituent molecules. Also seen in the micrographs were examples of a higher level of type XIX organization; a classic representative is shown in Fig. 9 (large panel). These featured clusters of the aggregates described above and comprised a great many individual type XIX molecules.
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To further ascertain the nature of the amino-terminal interactions, Western
blotting of purified type XIX was conducted under different conditions using
the NH2-Ab as probe (Fig.
10). As reported earlier for type XIX synthesized by RMS cells
(18), in the absence of
reducing agent, all of the intact type XIX molecules were found in the
stacking gel or at the top of the separating gel
(Fig. 10, lane 1). No
individual 165-kDa chains or lower molecular mass species were detected,
showing that the bands revealed in lanes 2 and 3 were a
function of the treatment indicated. Type XIX, digested with collagenase and
electrophoresed under nonreducing conditions, appeared mainly in the form of
several bands ranging in size from 110 to 
250 kDa
(Fig. 10, lane 2). A
band of 60 kDa was also seen. In the presence of DTT
(Fig. 10, lane 3),
the amino fragment migrated as a 34-kDa band (previously shown for
collagenase-treated, RMS type XIX)
(18). (The minor band of 29
kDa is due to a secondary cleavage site for collagenase.) The 110-kDa band (3
x 34 kDa) would represent the cleaved amino terminus of a monomer
(linked by interchain bonds), and the higher molecular weight bands correspond
to this domain in the oligomers. The intensity of the larger fragments will be
underrepresented compared with smaller ones due to the decrease in transfer
efficiency. Consistent with the electron microscopy images, these results
independently show that the type XIX molecules associate via their
amino-terminal domains and furthermore reveal that this complex occurs by
formation of intermolecular disulfide cross-links.
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The Type XIX Amino Terminus Contains a Heparin-binding Site—The type XIX amino terminus has been characterized as a Tsp-N module (10, 33, 34), and specific functions of thrombospondin-1 (TSP-1) have been mapped to this domain (reviewed in Refs. 35 and 36). Alignment of the modules in type XIX and TSP-1 revealed about 15% absolute identity as dispersed single residues or doublets (data not shown). The most conspicuous homology was the TSP-1 motif (RXXKKXR) (37) embedded in a longer basic residue sequence (RXRRXXKKXR) in type XIX (16). The KKXR peptide is a known TSP-1 heparin-binding site (38).
To ascertain whether this site in type XIX is functional, the SP Sepharose peak was digested with collagenase and applied to a heparin-Sepharose column. Western blotting demonstrated that all of the type XIX amino-terminal fragment bound to the column in 0.1 M NaCl, was retained after a 0.3 M NaCl wash, and eluted sharply in a 1 M NaCl buffer (Fig. 11). Prior chromatography showed that the fragment gradient-eluted at 0.70–0.75 M NaCl.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Type XIX Is a Rare Protein in Human Tissue—Type XIX is by
far the least abundant collagen so far purified, with a representation of
10–6% of the dry weight of umbilical cord
(even less than type VII collagen, which is 0.001% of pepsin-digested
skin tissue) (39). This value
should be reasonably accurate, since the relative amount of type XIX at each
stage was monitored to assess the recovery. Once optimized, the isolation
procedure was straightforward and designed to minimize loss by capitalizing on
favorable type XIX properties. Type XIX was readily extracted from tissue
using mild conditions, showing that it is not incorporated into an insoluble
matrix complex. At the next stage, an inefficient 5 M NaCl
precipitation was effectively replaced by use of 40% saturated ammonium
sulfate, and type XIX in the pelleted material was completely soluble in
neutral salt buffer. Strong affinity for a cation exchanger allowed for a
stringent wash and complete disassociation from the resin using a high salt
buffer. The rapid batch procedure was not replaced by gradient column elution,
since it was clear that ion exchange chromatography just served as a
prerequisite for specific capture by a type XIX antibody
(Fig. 3). The former step
afforded an 2000-fold enrichment and the latter an additional
7000-fold (totaling 1.4 x 107). The strategy
exemplified here (Fig. 5) may
provide a blueprint for purification of other extremely rare collagens. Only
the COOH-Ab bound to native type XIX, consistent with its ability to also
recognize the protein by immunohistochemistry
(18,
19). The NH2-Ab was
ineffective in both respects, probably due to inaccessibility of the epitope
within the clusters visualized by rotary shadowing.
Proteolysis Results in Discrete Type XIX Cleavage
Fragments—Two particularly labile sites resulted in distinct
cleavage products that were detected in Western blots and silver-stained gels
(Figs. 3 and
4). The type XIX
NH2-Ab reacted with a minor 120-kDa form that is probably generated
from cleavage in NC3, the 23-amino acid interruption (identified as flexible
site P5 in Fig. 12C)
separating COL3 from the uninterrupted COL2 subdomain. Although NC3 is 301
residues from the carboxyl terminus, this length corresponds to 45 kDa,
considering that the 1119-residue type XIX chain migrates as 165-kDa by
SDS-PAGE, 1.5 times the estimated size. (This retarded mobility is
characteristic of collagen chains.)
The 140-kDa degradation product, identified only by the COOH-Ab, increased
considerably after elution from the antibody column, where type XIX, at an
2 µg/ml concentration, is sensitive to even a trace amount of
protease. The cleavage is predicted to be near the junction of the 30-kDa
amino terminus (not to be confused with the 34-kDa collagenase-generated
fragment) and COL5 (see Fig.
12A). Accordingly, globular domains separated from the
kinked rods were prominent in rotary-shadowed images, and their shape was the
same as the corresponding regions in the intact molecule. Three lysines are
located within the last 14 residues of the type XIX amino terminus
(14); the middle one is a KD
sequence found at the equivalent position in the TSP-1 Tsp-N module
(37), where it is highly
susceptible to trypsin cleavage
(40). Identification of the
type XIX site will require a more abundant source of protein to obtain the
amount needed for sequencing. Characterization of these labile sites may prove
important in studies of type XIX catabolism and matrix remodeling.
The Type XIX Molecule Can Assume Many Conformations— Electron microscopy established that individual type XIX molecules span 240 nm, terminate with a small nodule at the amino end, and contain rodlike collagenous domains interrupted by sharp bends. The 220-nm length, estimated for the sum total of the collagenous rods, agrees well with the established value of 0.286 nm/residue (41); 712 residues x 0.286 = 204 nm, which would be a minimum, since the residues in NC5-2 could not be measured.
A wide array of different type XIX images was observed (Fig. 6, a–l). The histogram (Fig. 7) indicated that there are up to six highly flexible regions. Some type XIX molecules display all six kinks, whereas others have fewer; not all hinge regions are always apparent. There are examples of extended molecules with one or two kinks; these contrast with others that are highly bent and assume a "zigzag" shape (Fig. 6, a, e, k, and l). In some cases, the angles are so pronounced that a part of the molecule folds back on itself (Fig. 6, b, d, and g–j). Another flexible site may flank the amino terminus, where cleavage of exposed residues could give rise to the 140-kDa fragment; however, this remains unresolved, since a rod emanating from the nodule radially or tangentially cannot be easily differentiated.
A high correlation exists between the positions of the six major kinks and the internal NC segments. The five predicted type XIX collagenous subdomains are composed of 144, 224, 108, 168, and 70 amino acids (Fig. 12A), which would represent 20, 31, 15, 23.5, and 10%, respectively, of 220 nm. The length of each of these rods would therefore be 44, 68, 33, 52, and 23 nm, (discounting the NC5–2 segments), and the cumulative distance to the bends would be 64 (i.e. 44 + 20 nm), 132, 165, 217, and 240 nm (Fig. 12B). An illustration depicting the spatial relationship of the kinks (P1–P6 peaks in Fig. 7) to the type XIX domain structure reveals a remarkable coincidence of P2, P4, P5, and P6 with NC5, NC4, NC3, and NC2, respectively. The other two bends, P1 and P3, closely align with three-residue interruptions in COL5 and COL4. Extreme flexibility at such sites is not surprising, since in osteogenesis imperfecta even a single glycine substitution in type I collagen was shown to cause kinks (42–44).
The nonfibrillar collagens contain various size interruptions in the collagenous region; electron microscopy has been carried out to visualize some of these molecules (see Ref. 10 and references therein and Refs. 45–47). The kinks/bends that have been observed are often difficult to map to specific interruptions when the collagenous subdomains are not as defined as they are in type XIX and related family members (see Introduction). Alternatively, it is known that the presence of even large noncollagenous segments does not necessarily extrapolate into kinks as seen for the three-subdomain type IX collagenous region. Only one kink, equivalent to the 12–17-residue NC3 and the site of glycosaminoglycan chain attachment, is seen by electron microscopy despite the presence of a 30-residue NC2 segment (10, 48). In type XIX, manifestation of all of the large and at least several of the small interruptions into highly flexible sites results in the most polymorphic collagen so far characterized. The interruptions support numerous spatial configurations (illustrated in Fig. 12D) and, in this regard, can confer a high degree of adaptability to the microenvironment by differentially positioning the rigid collagenous subdomains. Moreover, the long, extensible NC segments may, in particular, represent binding sites whose availability can be modulated by the degree to which the residues are exposed to other matrix molecules. Taken together, the permutations in the type XIX structure are considerable, and even subtle differences may influence many biological processes.
The Supramolecular Assembly of Type XIX Is Dependent upon Amino-terminal Interactions—More than half the type XIX collagen molecules were present in oligomers interacting via their amino-terminal ends, suggesting that these complexes are physiologically significant. It is very unlikely that they could be attributed to a technical artifact, since the inherent low density of the type XIX molecules sprayed onto the surface never favored a crowded environment. There are many examples of self-assembly of biological systems, but structural proteins in particular are "built to assemble." In other words, the binding interactions that are required for organization of some biological structures (exemplified, in fact, by collagens) (8, 10) are often manifested in the purified protein(s), and therefore supramolecular aggregates observed by electron microscopy commonly reflect aspects of their in vivo assembly.
Nonfibrillar collagens, with most still to be characterized, are known to organize into diverse higher order structures (e.g. polygonal networks, hexagonal lattices, beaded filaments, antiparallel dimer filaments, and direct association with fibrillar collagens) (reviewed in Refs. 1, 2, 8, and 10). They occur through end-to-end and/or side-by-side interactions, and many, as shown here for type XIX, are stabilized by intermolecular disulfide cross-links. The type XIX arrangement represents a previously unknown structure. The only visual parallel is the type X collagen aggregates, which were found in cultured cells expressing the endogenous or recombinant protein (49, 50). The individual type X molecules radiate from a globular carboxyl-terminal (not amino-terminal) core, and these complexes appear to interconnect through antiparallel overlap of rodlike triple helices (8, 10, 49). As demonstrated in the type X studies (49, 50), immunoelectron microscopy and molecular aggregation using a recombinant form may prove useful to further elucidate the type XIX structure.
Potential Biological Significance of the Type XIX Amino
Terminus—Knowledge of the Tsp-N domain in other systems may provide
important clues about the role of the type XIX amino terminus. The Tsp-N
module is found in about 100 proteins, particularly those multidomain adhesive
proteins that act as molecular bridges between cells and matrix and
participate in cell-cell communication
(51). Tsp-N contains patterns
of alternating hydrophobicity characteristic of anti-parallel 
strands,
and the presence of the predicted sandwiches appears, at least in part, to
dictate the conserved, known and predicted, structural homology
(34). A high propensity for
sheet formation is also found in the Tsp-N/amino terminus of type XIX
(14). Functions ascribed to
Tsp-N of TSP-1 include cell adhesion, spreading, and migration; binding to
glycosaminoglycan chains; disruption of focal contacts; regulation of
proliferation; endocytosis; and platelet aggregation
(35). Statistical analysis
corroborated by the crystal structure has also shown a similarity of Tsp-N to
the laminin G module, which shares a number of the aforementioned activities
in addition to signaling, assembly, and differentiation
(51,
52). The Tsp-N module is found
too in TSP-like NELL proteins that are involved in neurogenesis and the
maintenance of neuronal plasticity
(53).
The Tsp-N module is also present in collagens IX, XII, XIV, and XVI (10, 17, 33, 34), but the KKXR heparin-binding site in TSP-1 is only conserved in type XIX. It has been shown that TSP-1, mediated by the Tsp-N module, binds to cells and is internalized and degraded in a process that requires heparan sulfate proteoglycans (54–56). One can speculate that the type XIX oligomers may function as a nidus to increase the local concentration of signaling/structural molecules by associating with multiple extracellular matrix heparan sulfate proteoglycans, and/or type XIX could serve as a cell-cell/cell-matrix adhesive protein by binding to transmembrane heparan sulfate proteoglycans, like the syndecans, as has been established for TSP-1 (35, 36). Taken together, the results shown here provide a solid foundation for approaching functional studies of type XIX in the basement membrane/zone and in myogenic processes.
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FOOTNOTES |
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To whom correspondence should be addressed: University of Pennsylvania School
of Medicine, Dept. of Biochemistry and Biophysics, 909 Stellar Chance, 422
Curie Blvd., Philadelphia, PA 19104-6059. Tel.: 215-898-0712; Fax:
215-573-2085; E-mail:
myers{at}mail.med.upenn.edu.
1 The abbreviations used are: RMS, rhabdomyosarcoma; Tsp-N,
thrombospondin-amino terminal domain; BMZ, basement membrane zone; COOH-Ab,
type XIX antibody recognizing the noncollagenous carboxyl peptide;
NH2-Ab, type XIX antibody recognizing the amino-terminal
noncollagenous domain; TSP-1, thrombospondin-1; NC, noncollagenous; COL,
collagenous subdomain; DTT, dithiothreitol.
2 F. Ramirez, personal communication.
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