(Received for publication, February 11, 1997, and in revised form, April 28, 1997)
From the Departments of Biochemistry and Molecular Biology, and
Anatomy and Cell Biology, University of Kansas Medical
Center, Kansas City, Kansas 66160-7421, the § Department of
Nephrology, University Hospital, S-221 85 Lund, Sweden, the
¶ Penn Center for Molecular Studies of Kidney Diseases, Renal
Electrolyte and Hypertension, University of Pennsylvania Medical
School, Philadelphia, Pennsylvania 19104, and the
Renal
Division, Department of Medicine, Brigham and Women's Hospital,
Harvard Medical School, Boston, Massachusetts 02115
Seminiferous tubule basement membrane (STBM)
plays an important role in spermatogenesis. In the present study, the
composition and structural organization of type IV collagen of bovine
STBM was investigated. STBM was found to be composed of all six
-chains of type IV collagen based upon immunocytochemical and
biochemical analysis. The content of
3(IV) chain (40%) and the
4(IV) chain (18%) was substantially higher than in any other
basement membrane collagen. The supramolecular structure of the six
(IV) chains was investigated using pseudolysin (EC 3.4.24.26)
digestion to excise triple-helical molecules, subsequent collagenase
digestion to produce NC1 hexamers and antibody affinity chromatography
to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of
(IV) chains, were characterized for their
(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with
(IV) chain-specific antibodies. Three major hexamer populations were found
that represent the classical network of the
1(IV) and
2(IV) chains and two novel networks, one composed of the
1(IV)-
6(IV) chains and the other composed of the
3(IV)-
6(IV) chains. The results establish a structural linkage between the
3(IV) and
5(IV) chains, suggesting a molecular basis for the conundrum in
which mutations in the gene encoding the
5(IV) chain cause defective
assembly of the
3(IV) chain in the glomerular basement membrane of
patients with Alport syndrome.
Basement membranes (BMs)1 are thin sheet-like extracellular structures that compartmentalize tissues. They are substrata for cells of various organs and provide important signals for differentiation, maintenance, and remodeling of tissues. BM function is altered in acquired and genetic diseases, such as Goodpasture syndrome, an autoimmune disorder, Alport syndrome, a form of hereditary nephritis, and diffuse leiomyomatosis, a hereditary disease characterized by benign proliferation of smooth muscle. Type IV collagen, the major constituent of BMs has been linked to the pathogenesis of each of these disorders (1).
Type IV collagen has recently emerged as a family of triple-helical
isoforms consisting of six genetically-distinct chains, designated
1(IV) to
6(IV) (1-8). The entire coding sequences for all six
human
(IV) chains and certain
(IV) chains from other species have
now been determined (2-22). Their primary structures are similar. Each
is characterized by a ~25-residue noncollagenous sequence at the
amino terminus, a ~230-residue noncollagenous (NC1) sequence at the
carboxyl terminus, and, between these sequences, a long collagenous
domain of ~1400 residues of Gly-Xaa-Yaa repeats that, together with
two other
(IV) chains, forms the triple helix. The first ~130
residues of the collagenous domain is called the 7 S domain and is
involved in tetramerization of triple helical protomers, whereas the
NC1 domain is involved in their dimerization. The collagenous domain is
interrupted by more than 20 short nontriple-helical regions which are
thought to increase flexibility of this collagenous region. Analysis of
the
1(IV) and
2(IV) chains reveals that the Gly-X-Y
sequences flanking these interruptions are atypical when compared with
the remainder of the collagenous domain. The atypical flanking
sequences may be important in stabilizing the triple helix and in the
formation of polygonal networks in BMs (23).
The existence of six (IV) chains allows for as many as 56 different
kinds (isoforms) of triple-helical molecules, which differ in type and
stoichiometry of chains. Evidence has been obtained for heterotrimers
that have chain composition of [
1(IV)]2
2(IV) and
[
3(IV)]2
4(IV) (24, 25). Whether the
5(IV) and
6(IV) chains occur in some combination with
1(IV) to
4(IV) or
in separate molecules remains unknown.
The six (IV) chains differ considerably with respect to tissue
distribution. At the protein level, immunochemical studies have shown
that the
1(IV) and
2(IV) chains have a ubiquitous distribution
whereas the
3(IV),
4(IV), and
5(IV) chains have a restricted
distribution in both human and rodent tissues (26-29). At the mRNA
level, the relative expression of the
3(IV),
4(IV),
5(IV), and
6(IV) chains varies greatly among a variety of human tissues,
including a variation in the ratio of expression of the
3(IV) and
4(IV) chains (16, 17). Thus, the chain composition of a BM may be
tissue specific. Likewise, the kind of triple-helical isoform(s) and
their supramolecular organization in BM may be tissue-specific.
The 3(IV),
4(IV),
5(IV), and
6(IV) chains have all been
implicated in the pathogenesis of human diseases (1, 7, 30-33). In
Goodpasture syndrome, the
3(IV) chain is the target for the
pathogenic autoantibodies. In Alport syndrome, the COL4A5 gene encoding
the
5(IV) chain is mutated in the common X-linked form of the
disease, and the COL4A3 and COL4A4 genes are mutated in the autosomal
recessive form (1). In leiomyomatosis, the COL4A5 and COL4A6 genes are
deleted in some patients. These mutations together with the restricted
tissue expression of chains indicate specific biological functions of
the
3(IV),
4(IV),
5(IV), and
6(IV) chains, including a
vital role in the molecular sieve function of the glomerular BM and in
differentiation of smooth muscle cells.
Knowledge of the tissue-specific composition and organization of
(IV) chains of several distinct BMs is of fundamental importance for
elucidating the structure/function relationships of these chains and
their role in mechanisms underlying diseases. To date, studies on
organization have focused primarily on the BM of the renal glomerulus,
owing to the well established role of glomerular BM in the molecular
sieve function of the kidney and the loss of this function in
Goodpasture and Alport syndromes. In the present study, the composition
and organization of the
(IV) chains of seminiferous tubule BM (STBM)
was investigated. STBM was chosen for study because it appears to have
a special role in spermatogenesis, ultrastructural abnormalities are
thought to lead to infertility, and temporal studies of the expression
of type IV collagen chains show the unique expression of the
3(IV)
chain at the initiation of spermatogenesis (34). The results
reveal that STBM contains the highest percentage of the
3(IV) chain
of any BM collagen thus far studied and that it exists in a novel
supramolecular complex comprised of the
3(IV),
4(IV),
5(IV), and
6(IV) chains.
Frozen bovine testes were purchased from
Pel-Freez Biological (Rogers, AR) and stored at 20 °C. Bacterial
collagenase was obtained from Worthington Biochemical (Freehold, NJ).
DE52-cellulose was obtained from Whatman (Hillsboro, OR), Sephacryl
S-300 and Sephacryl S-200 from Pharmacia Biotech Inc. DNase and the
protease inhibitors, N-ethylmaleimide, EDTA, and
6-aminohexanoic acid, were obtained from Sigma or Peninsula
Laboratories (Belmont, CA). The C18 reversed-phase HPLC
column (201 TP 104, 10 µm) was purchased from Vydac (Hesperia, CA).
Reagents for polyacrylamide gel electrophoresis were from Bio-Rad.
Pre-stained protein markers were obtained from Life Technologies, Inc.
All secondary antibody conjugates were obtained from Dakopatt
(Denmark). Pseudolysin2 (EC 3.4.24.26) (35)
was purchased from Nagase Biochemical, Fukushiyama, Japan.
The antisera used for primary antibody were either
from patients with Goodpasture syndrome (autoantiserum) and Alport
syndrome (alloantiserum) both directed against 3(IV) NC1 (36, 37) or
from rabbits immunized with the monomeric subunits of
1(IV) and
2(IV) NCI domain of type IV collagen (38) or anti-
3(IV) (38),
anti-
4(IV) (38), anti-
5(IV) (39), or anti-
6(IV) (29) synthetic
peptide antisera. The secondary antibody used in immunocytochemistry
and immunoblotting was either rabbit anti-human IgG or swine
anti-rabbit IgG conjugated to horseradish peroxidase.
Fresh bovine testes were obtained at a slaughterhouse and
quickly frozen on dry ice. Cryostat sections of bovine testis were placed on polylysine-coated slides. The sections were fixed for 10-20
min with cold acetone. To gain access to antigenic epitopes, the
cryostat sections were washed in 0.15 M NaCl, 10 mM sodium phosphate, pH 7.4, and then treated with 6 M urea, 0.1 M glycine-HCl, pH 3.6, for 1 h
at room temperature. The sections were washed in 0.15 M
NaCl, 10 mM sodium phosphate, pH 7.4, and then blocked with
1:1 Blotto (40):10% normal goat or rabbit serum plus 0.1% protease
inhibitor mixture (41), 0.1% Tween 20 in 0.15 M NaCl, 10 mM sodium phosphate, pH 7.4, for 20 min at room
temperature. Primary antibodies were added for 2 h at room
temperature. Normal serum of the same species as the secondary antibody
was added at 2% along with 0.1% Tween 20 to the diluted primary
antibody. Primary antibodies were rabbit anti-bovine 1(IV)/
2(IV)
NC1 (diluted 1/100 to 1/500), Alport syndrome alloantibodies eluted
from a rejected transplanted kidney (36) (diluted 1/500 to 1/1000), and
Goodpasture syndrome antibody (37) (diluted 1/50 to 1/100). Anti-peptide antisera specific for
3(IV),
4(IV),
5(IV), and
6(IV) NC1 were used at a dilution of 1/150 to 1/500. The sections were washed three times in 0.15 M NaCl, 10 mM
sodium phosphate, pH 7.4, and then incubated in peroxidase-labeled
goat-anti rabbit IgG (Sigma; diluted 1/1000 to 1/2500) or rabbit-anti
human IgG (diluted 1/2000) for 2 h at room temperature. Again 2%
normal serum was added to reduce nonspecific binding. The sections were washed three times in 0.15 M NaCl, 10 mM sodium
phosphate, pH 7.4. Diaminobenzidine was used to develop a brown
reaction product. The sections were then lightly counterstained with
hematoxylin. Controls included the substitution of normal rabbit serum
for anti-
1(IV)/
2(IV), or the use of no primary antibody instead of Alport syndrome alloantibodies. Sections were examined with a Zeiss
Axioskop and photographs made with Kodak Ektar 25 film using an 80A
filter.
The protocol for preparing STBM sleeves was as described by Enders et al. (41), with minor modifications. Testes were decapsulated, ground with a meat grinder, and blended to a liquified state with distilled water, 0.05% sodium azide and then shaken for 1 h on ice and pelleted by centrifugation at 8,000 × g for 10 min. The pellet was washed three times with the same solution and then suspended in 1 M NaCl containing 200 Kunitz units/ml DNase, 2 mM phenylmethylsulfonyl fluoride, 0.1% protease inhibitors (42) and gently shaken for 90 min at room temperature. The solution was pelleted by centrifugation at 8,000 × g for 10 min and the pellet resuspended in 1% sodium deoxycholate and gently shaken for 1 h on ice. The resulting pellet was washed three times with distilled water.
Digestion of STBM with Pseudomonas aeruginosa PseudolysinSTBM (1 g) was digested with 0.5% pseudolysin at 4 °C for 24 h, and the reaction was arrested by the addition of 20 mM EDTA as described previously (38). The pseudolysin-soluble fraction was separated from enzyme and EDTA by twice precipitating with 20% NaCl. About 20-30 mg of the resulting precipitate was dissolved in 0.15 M NaCl, 50 mM Tris-HCl, pH 7.5, and fractionated on a Sephacryl S-1000 gel filtration column equilibrated with the same buffer to remove residual amounts of pseudolysin from type IV collagen components. The soluble fraction represented about 16% of the STBM. Both pseudolysin-soluble and pseudolysin-insoluble fractions of STBM were used for further study.
Electron MicroscopyThe pseudolysin soluble STBM (20 µg/ml) was sprayed in 0.15 M ammonium bicarbonate, 50% glycerol or 0.05 M acetic acid, 50% glycerol and further treated for rotary shadowing following established procedures (43). Samples were examined using a JOEL JEM-100CX II electron microscope.
Isolation of NC1 HexamerNC1 hexamer was prepared as described previously (44). STBM, pseudolysin-soluble STBM, or pseudolysin-insoluble STBM was suspended in collagenase buffer (25 mM 6-aminohexanoic acid, 10 mM CaCl2, 5 mM benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride, 50 mM HEPES, pH 7.5). Bacterial collagenase (0.2% w/w) was added and the suspension stirred continuously at 37 °C for 24 h, followed by addition of 20 mM EDTA to stop the digestion. The supernatant solution, containing the solubilized NC1 hexamer, was dialyzed against 50 mM Tris-HCl buffer, pH 7.5, and applied to a DE52-cellulose anion exchange column (2.5 × 35 cm) already equilibrated with the dialysis buffer. The unbound material, which contained the 7 S domain and NC1 hexamer, was concentrated in an ultrafiltration cell (YM 10 filters, Amicon) and fractionated on a Sephacryl S-300 column (2.2 × 81 cm), equilibrated with 0.15 M NaCl, 0.02% sodium azide, 50 mM Tris-HCl, pH 7.5. The fractions containing the NC1 hexamer were pooled and used for subsequent studies.
Electrophoretic AnalysisOne-dimensional SDS-PAGE was performed using the discontinuous buffer system described by Laemmli (45) in a 4-22% gel gradient. For two-dimensional gel electrophoresis, the first dimension was performed according to O'Farrell (46), with some modifications (43), and the second dimension was SDS-PAGE using a 10-22% gel gradient. Samples for electrophoretic analysis were either lyophilized or ethanol precipitated when necessary. Gels were either stained with Coomassie Brilliant Blue R-250 or silver as described by Morrissey (47).
Amino Acid Sequence AnalysisAmino-terminal amino acid
sequence analysis for 3(IV) NC1 monomer was performed at the
University of Kansas Medical Center Biotechnology Support Facility. A
lyophilized
3(IV) NC1 monomer preparation was purified by
C18 reversed-phase HPLC, derivatized with
phenylthiohydantoin, and then identified by use of a 470A protein
sequencer with an on-line 120A PTH analyzer.
To perform immunoblotting, the SDS-PAGE separated proteins were transferred electrophoretically to nitrocellulose paper as described previously (43), then blocked with bovine serum albumin and reacted with primary and secondary antibodies by methods previously described (48).
Chromatographic TechniquesQuantitation of hexamer subunits was performed on a C18 reversed-phase HPLC column as described previously (43). The identification of subunits in different pools from HPLC was performed by silver staining of two-dimensional electrophoretic gels and by immunoblotting with chain-specific antibodies.
Affinity ChromatographyTwo affinity chromatographic
columns were prepared using monoclonal antibodies mAb6 and mAb17,
specific for 1(IV) NC1 and
3(IV) NC1, respectively (25). NC1
hexamers contained in the various affinity column fractions were
further analyzed by C18 reversed-phase HPLC as described
previously (43).
Immunocytochemical Localization of Type IV Collagen Chains in Bovine STBM
The localization of type IV collagen chains in testis tissue was
determined using chain-specific antibodies (Fig. 1). The 1(IV)/
2(IV) chains were localized to STBM (Fig. 1, A
and B, large filled arrow) and the interstitial
extracellular matrices, including blood vessels (Fig. 1B, open
arrowhead). When normal rabbit serum was substituted for
anti-
l(IV)/
2(IV) antisera, no specific localization was detected
(Fig. 1A). In contrast, the
3(IV) chain was localized to
the STBM using Alport alloantibody or Goodpasture autoantibody (Fig.
1C) both of which are directed against the
3(IV) chain
(3, 36, 37) and using antipeptide antisera to
3(IV) NC1 (Fig.
1E). Similarly, the
4(IV) and
5(IV) chains were
localized to STBM (Fig. 1, F and G). The
6(IV)
chain was localized to the STBM and larger blood vessel BM (Fig.
1H). Thus, the
3(IV),
4(IV), and
5(IV) chains are
exclusively localized to the STBM and the STBM is comprised of all six
(IV) chains.
Chain Composition of Type IV Collagen
The kind and relative amount of type IV collagen chains in STBM were determined by quantitative analysis of their NC1 domains. Previous studies established that the respective NC1 domains, in a hexameric configuration can be obtained from basement membrane by collagenase digestion, identified by two-dimensional electrophoresis using chain specific antibodies for immunoblots, and resolved and quantitated by C18 reversed-phase HPLC (43).
The NC1 hexamer of STBM was purified under nondenaturing conditions by
ion-exchange and gel filtration chromatography and then characterized
by a variety of approaches. The hexamer eluted from a Sephracryl S-300
column (Fig. 2A) in a position identical to
that of NC1 hexamer from glomerular BM (43). Pool II was examined by
rotary shadowing electron microscopy to confirm the integrity of the
hexamers (Fig. 2B). The yield of hexamer was 6-7 mg/g dry
weight STBM. In the presence of 6 M guanidine-HCl (Fig.
2C) or 10% SDS (Fig. 2D), the NC1 hexamer
dissociated into monomer and dimer subunits. The relative amounts of
dimer and monomer were determined from the elution profile (Fig.
2C) to be 60 and 40%, respectively. Upon reduction of
disulfide bonds in the presence of SDS, dimers completely dissociated
into monomers (Fig. 2D).
STBM-NC1 hexamer was analyzed both by two-dimensional electrophoresis
and by C18 reversed-phase HPLC to identify and quantitate the kinds of NC1 domain subunits. The electrophoresis pattern revealed
a complex set of >20 dimers and >10 monomers (Fig.
3A). The pattern is qualitatively similar to
that of the NCl hexamer from glomerular BM (43), but differs in the
relative abundance of the subunits. STBM-NC1 hexamer was dissociated
with 0.1% trifluoroacetic acid and the subunits were resolved by HPLC
into four fractions (I-IV) (Fig. 3B). NC1 subunits
comprising each fraction were identified by two-dimensional
electrophoresis on the basis of: 1) their migration positions (Fig.
4, A-D) in relation to that previously
established for NC1 subunits of bovine glomerular BM (43); and 2) their binding to chain-specific antibodies (Fig. 4, E-L). Fraction
I contained 1(IV),
2(IV), and
5(IV) NC1 monomers; fraction II contained
1(IV),
2(IV), and
5(IV) NC1 dimers; fraction III contained
4(IV) NC1 monomers and dimers and
6(IV) NC1 dimers; and
fraction IV contained
3(IV) NC1 monomers and dimers and trace amounts of
5(IV) NC1. In each case, dimers have apparent size isoforms, and both monomers and dimers have charge isoforms. The identity of the
3(IV) NC1 monomers, designated
3aNC1 and
3bNC1 in Fig. 4D, was also determined by amino-terminal sequence
analysis. The sequence of the first 12 residues of
3aNC1 and
3bNC1 was identical to that previously established for the
3(IV)
subunits of bovine glomerular BM (4). Only subunits comprising fraction IV bound Goodpasture autoantibodies and Alport alloantibodies reflecting the presence of
3(IV) NC1 domain as their target as previously established for GBM (3, 36, 37). The relative amount of
1(IV),
2(IV),
3(IV), and
4(IV) NCl subunits comprising STBM-NCl hexamer was determined from the HPLC profile (Fig.
3B). The relative amount of
5(IV) NC1 subunit was
obtained from affinity chromatography experiments and reversed-phase
HPLC (see below). The results (Table I) reveal that STBM
has the highest proportion of
3(IV) (40%) and
4(IV) NCl domains
(18%) of all mammalian BMs for which quantities have been
reported.
|
Organization of Type IV Collagen in STBM
Pseudolysin-soluble STBMSTBM was digested with pseudolysin
at 4 °C for 24 h, which was previously established to
solubilize truncated protomers of type IV collagen that retain a
portion of the triple-helical domain and the complete NC1 domain (38).
These truncated molecules were characterized by rotary shadowing
electron microscopy (Fig. 5). They have a triple-helical
(rod-like) segment, 287 nm in length, linked to a globular NC1 domain
and are dimerized through NC1-NC1 interactions forming molecules with
lengths of approximately 600 nm. The identity of chains comprising
these molecules was determined by analysis of respective NC1 domains
that were released upon collagenase digestion. The identity of the
NC1 domain was determined by HPLC chromatography and
two-dimensional gel electrophoresis and immunoblotting (Fig.
6) on the basis of the identity of the components
established in Fig. 4B. The two-dimensional immunoblotting pattern indicated that the NC1 domain contains 1(IV) and
2(IV) NC1 monomers and dimers. The HPLC profile (panel A) supports
this identity and shows that
1(IV) and
2(IV) NC1 domains comprise >95% of the NC1 domains, reflecting that the triple-helical molecules shown in Fig. 5 are comprised of
1(IV) and
2(IV) chains.
To determine which chains are connected through NC1-NC1 interactions,
NC1 hexamers3 released from the truncated
molecules by collagenase digestion were fractionated using two
monoclonal affinity columns (anti-1(IV) NC1 and anti-
2(IV) NC1)
using a strategy previously described (3, 25). When the NC1 hexamer was
applied to an anti-
3(IV) NC1 affinity column (Fig.
7A), about 10% bound to the column. The
chain identity of the respective NC1 domains was determined by
two-dimensional gel electrophoresis (Fig. 7B) and
immunoblotting (Fig. 7, C-G). The results revealed that the
bound hexamer contained monomers of
1(IV),
2(IV),
3(IV), and
4(IV) NC1 domains and homodimers of
1(IV),
3(IV), and
4(IV)
NC1 domains, but no
2(IV) NC1 dimer. Thus, this population(s) of NC1
hexamer is comprised of
3(IV) NC1 in association with
1(IV),
2(IV), and
4(IV) NC1. It represents 2% of the total NC1 hexamer
of STBM and it is designated hexamer population A.
The unbound fraction was then applied to the anti-1(IV) affinity
column and all of the sample bound to the column (Fig.
8A). The bound hexamer was analyzed by
two-dimensional gel electrophoresis. The pattern of the two-dimensional
spots was similar to that of the NCl hexamer composed of
1(IV) and
2(IV) NCl (Fig. 8B) in the immunoblot analysis.
Furthermore, the bound fraction showed strong reactivity with the
antibodies to
1(IV) and
2(IV) NC1 domain (Fig. 8C),
but not with the antibodies to
3(IV),
4(IV),
5(IV), and
6(IV)NCl (data not shown). Therefore, this NCl hexamer population,
which did not bind to the anti-
3(IV) affinity column but bound to
the anti-
1(IV) affinity column, contained only
1(IV) and
2(IV)
chains. Thus, this population(s) of NCl hexamer A (Fig. 11) is composed
of
1(IV) NC1 associated with an
2(IV) NC1. It represents 14% of
the total NC1 hexamer of STBM and is designated hexamer population
B.
Pseudolysin-insoluble STBM
The identity and organization of
chains connected by NC1-NC1 interactions that comprise the
pseudolysin-insoluble STBM was determined by analysis of the NC1
domains released upon collagenase digestion as described (see above)
for the soluble STBM fraction. Insoluble STBM is comprised of 85%
3(IV),
4(IV),
5(IV), and
6(IV) NC1 monomers and dimers, and
the remainder (<15%) of
1(IV) and
2(IV) NC1 monomers and dimers
(Fig. 6, C and D).
The NC1 hexamer populations comprising this insoluble STBM were
fractionated using the two affinity columns and the hexamer characterized by two-dimensional gel electrophoresis as was done for
the soluble STBM (see above). When the NC1 hexamer was applied to the
anti-1(IV)NCl affinity column, about 45% of the protein bound to
the column (Fig. 9A). The bound fraction was
analyzed by two-dimensional gel electrophoresis and the pattern of the spots was essentially identical to that of the whole NCl hexamer (Fig.
3). Furthermore, the two-dimensional immunoblots showed that the bound
fraction reacted with antibodies to
1/
2(IV),
3(IV),
4(IV),
5(IV), and
6(IV)NCl at the monomer and dimer regions (Fig. 9,
C-G). Thus, the NC1 hexamer that bound to the anti-
1(IV)
affinity column bound NCl hexamer contained all six
(IV) chains. It
is composed of
1(IV) in association with
2(IV),
3(IV),
4(IV),
5(IV), and
6(IV) NC1 domains. It represents 35% of the
total NC1 hexamer of STBM and is designated hexamer population
C.
When the unbound fraction from the anti-1(IV) NCl affinity column
was applied to the anti-
3(IV) NCl affinity column, about 90% of the
total protein bound to the column (Fig.
10A). The two-dimensional gel
electrophoresis (Fig. 10B) and the two-dimensional
immunoblot (Fig. 10, C-G) analysis revealed the presence of
3(IV) NCl-
6(IV) NCl domains and the absence of
1(IV) and
2(IV) NC1 domains. Thus, this population(s) of NCl hexamer is
composed of
3(IV) NC1 domains in association with
4(IV),
5(IV),
6(IV) NC1 domains. It represents 49% of the total STBM
and is designated population D (Fig. 11).
Analysis of this population by reversed-phase HPLC revealed it to be
comprised of 66%
3(IV) NC1, 27%
4(IV) NC1 and
6(IV) NC1, and
7%
5(IV) NC1. The
4(IV) content represents the majority of the
27% of
4(IV) plus
6(IV) NC1 domains because
6(IV) NC1
represents only a small fraction, as is shown by the relative
abundance of spots (Fig. 4).
STBM was found to be composed of all six -chains of type IV
collagen. The evidence was based upon immunocytochemical analysis of
testis tissue and biochemical analysis of the NC1 domain hexamer that
was released upon collagenase digestion of STBM. The
3(IV),
4(IV),
5(IV), and
6(IV) chains are usually regarded as the minor chains of BMs in relation to the abundant
1(IV) and
2(IV) chains. However, in the case of STBM, the
3(IV)-
6(IV) chains collectively represent more than 80% of the chains, with the
3(IV) and
4(IV) chains as the major components. These results are in agreement with the supposition that BMs have a tissue-specific composition and organization of type IV collagen.
Pseudolysin cleavage of STBM was used to excise soluble triple helical
molecules for determination of their chain organization, as reflected
by compositions of NC1 hexamers that were subsequently released upon
digestion with collagenase. Four distinct populations of NC1 hexamers
were found, A and B from pseudolysin-soluble and C and D from pseudolysin-insoluble STBM (Fig.
11). Analysis of populations A-D leads to several
conclusions about the organization of the six (IV) collagen chains.
First, in population A, in an individual hexamer, one chain
of the two triple-helical isoforms (connected through their NC1
domains) is
3(IV) and the remaining five chains could be
1(IV),
2(IV),
3(IV), or
4(IV), or a combination of these chains. The
existence of an
3(IV) homohexamer cannot be dismissed, but a
homohexamer of
1(IV),
2(IV), or
4(IV) can be. Second, in
population B, in an individual hexamer, one chain of the two
triple-helical isoforms is
1(IV) and the remaining five chains could
be
1(IV) or
2(IV) or a combination of
1(IV) and
2(IV)
chains. The existence of an
1(IV) homohexamer cannot be dismissed,
but a homohexamer of
2(IV) can be. Third, in population C, in an individual hexamer, one chain of the two-triple
helical isoforms is
1(IV) and the remaining five chains could be
either
1(IV),
2(IV),
3(IV),
4(IV),
5(IV), or
6(IV) or
a combination of these chains. The existence of an
1(IV) homohexamer
cannot be dismissed, but a homohexamer of
2(IV),
3(IV),
4(IV),
5(IV), and
6(IV) can be. Fourth, in population D, in
an individual hexamer, one chain of the two triple helical isoforms is
3(IV) and the remaining five chains could be either
3(IV),
4(IV),
5(IV), or
6(IV) or a combination of these chains. The
existence of an
3(IV) homohexamer cannot be dismissed, but a
homohexamer of
4(IV),
5(IV), and
6(IV) can be. Fifth, the
composition of populations A-D clearly reveals that the six
(IV) chains have a limited number of ways in which they associate to
form dimers of triple-helical isoforms. This is because a random
distribution would result in the most abundant hexamer population
containing all six kinds of NC1 domains instead of the
3(IV),
4(IV),
5(IV), and
6(IV) NC1 domains observed (population
D, Fig. 11). Sixth, populations C and
D represent novel arrangements of chains, forming triple helical isoforms and supramolecular structures.
The exact number of isoforms of collagen IV is not known. There are 56 possible isoforms if the six (IV) chains can assemble randomly, and
18 if they assemble analogously to the well characterized [
1(IV)]2
2(IV) isoform from the EHS matrix (24),
wherein an
3(IV) or
5(IV) chain can replace an
1(IV) chain and
an
4(IV) or
6(IV) chain can replace
2(IV) chain. In these
configurations, the isoforms would have the generic composition:
[
1(IV)-like]2
2(IV)-like, relating to their primary
structures which are categorized as
1(IV)-like and
2(IV)-like.
Clearly, population B is consistent with the
[
1(IV)]2
2(IV) isoform. The composition of
population D provides the first evidence for an
[
3(IV)]2
4(IV) isoform devoid of
5(IV) and
6(IV) chains, as well as an isoform that connects an
3(IV) chain
with an
5(IV), either within a triple helix or between adjoining
triple helices.4 The possible isoforms in
population D, which are dimerized through NC1 domains, are:
[
3(IV)]2
4(IV),
3(IV)
4(IV)
5(IV),
3(IV)
5(IV)
6(IV), and [
5(IV)]2
6(IV). In
addition to these putative isoforms and the
[
1(IV)]2
2(IV) isoform, the chain content of
populations A and C is consistent with the
presence of [
1(IV)]2
4(IV),
[
1(IV)]2
6(IV), [
3(IV)]2
6(IV),
1(IV)
2(IV)
3(IV),
1(IV)
2(IV)
5(IV),
1(IV)
3(IV)
4(IV), and
1(IV)
3(IV)
6(IV) isoforms. Thus, the affinity chromatography results suggest up to 12 putative isoforms. However, for simplicity, the remainder of the discussion is based on the four isoforms: [
1(IV)]2
2(IV), [
3(IV)]2
4(IV),
3(IV)
4(IV)
5(IV), and
3(IV)
5(IV)
6(IV); this will not
affect the qualitative interpretation of the results. Accordingly, the
simplest interpretation of the affinity chromatography results is that
there are seven dimeric species of isoform in the supramolecular
structure of STBM collagen IV (Fig. 12, panel A); these dimers are designated NC1-linked
isoforms.
NC1 hexamer population B reflects the classical organization
in which the 1(IV) chain is linked to the
2(IV) chain. Its precursor is a pair of truncated protomers linked by NC1 domains (Fig.
5). It is similar in these respects to collagen IV from mouse EHS tumor
matrix (49), which has a well characterized net-like supramolecular
structure (reviewed in Ref. 50). There is published evidence that
isoforms containing
1(IV) and
2(IV) chains can form an
independent network in tissues that also contain the
3(IV)-
6(IV)
chains. Immunolocalization studies of the kidney show that only
1(IV) and
2(IV) chains are found in the mesangium and artery,
whereas chains
1(IV) through
5(IV) are found in glomerular and
tubular BM (26-28). Thus, isoforms containing
1(IV) and
2(IV)
chains must form an independent network in the kidney mesangium and artery, and perhaps form an independent network in
glomerular and tubular BM. Further, circumstantial, support for an
independent
1(IV)/
2(IV) network is that these chains appear to
have a ubiquitous occurrence in tissues, in contrast to the restricted
distribution of the other
(IV) chains (1). In STBM, hexamer
population B contains NC1 domains derived only from
1(IV)
and
2(IV) chains. These results are consistent with an independent
network that is composed of
1(IV) and
2(IV) chains. A portion of
this hypothetical network is portrayed in Fig. 12, panel
B.
NC1 hexamer population C reflects a novel organization in
which the 1(IV) chain is linked to
2(IV)-
6(IV) chains,
suggesting a network that is composed of all six
(IV) chains. A
portion of this hypothetical network is portrayed in Fig. 12,
panel C. These results corroborate the immunocytochemical
findings (Fig. 1) that all six
(IV) chains exist in STBM. The
possibility of this complicated type of network had been realized when
the
3(IV) and
4(IV) chains were discovered (51) and our results
provide evidence for it in STBM. Similar results for glomerular BM
provide evidence for it in that membrane too (3, 25).
NC1 hexamer population D reflects a novel organization in
which the 3(IV) chain is linked to the
4(IV),
5(IV), and
6(IV) chains. At one extreme, the
3(IV) chain could be linked to
five
4(IV) chains in the hexamer, forming two triple-helical
isoforms that are connected through their respective NC1 domains, or to five
5(IV) chains or five
6(IV) chains. On the other extreme, the
3(IV) chain could be linked to five chains in the hexamer in which
the
4(IV),
5(IV), and
6(IV) chain are components. In either
extreme, population D links an
3(IV) chain with an
5(IV) chain. The results are consistent with a separate network composed of only
3(IV),
4(IV),
5(IV), and
6(IV) chains, as portrayed in Fig. 12, panel D.
In summary, the three major populations B, C, and
D suggest the existence of three distinct collagen IV
networks. However, all three could be connected into one supra-complex
through association of 7 S domains, i.e. tetramerization of
the amino-terminal domain, thus forming a single network. If all three
putative networks are, instead, part of a single network, then they
would represent regions of that network. In addition, all six (IV)
chains contain cysteine residues in their collagenous domains. This
introduces another level of complexity in the STBM collagen
supramolecular structure because it is possible that the various
isoforms are cross-linked through disulfide bonds.
The structural linkage between an 3(IV) chain and an
5(IV) chain,
as revealed by hexamer population D, is particularly
noteworthy in view of the well described conundrum of the abnormality
that occurs in patients with X-linked Alport syndrome. In these cases, mutations in the COL4A5 gene, encoding the
5(IV) chain, cause defective assembly of the
3(IV) chain in the glomerular BM (1, 29,
36, 52, 53). A structural linkage between the
3(IV) and
5(IV)
chains suggests that the
5(IV) chain may be required for the
incorporation of the
3(IV) chain into a triple-helical isoform
containing both
3(IV) and
5(IV) chains or into two triple-helical isoforms, connected through NC1 interactions, in which the
3(IV) chain is contained within one isoform and the
5(IV) chain in the
other. The dependence of one chain on the assembly of another chain is
well established in the case of osteogenesis imperfecta in which
mutations in the
1(I) chain cause defective incorporation of the
2(I) chain (54).
The structural linkage between the 3(IV) and
5(IV) chains in STBM
raises questions about the molecular and fertility consequences of
COL4A5 gene mutations in patients with Alport syndrome. The STBM serves
several functions, including: (a) the site for
spermatogonial proliferation, (b) support for the phenotypic
differentiation of Sertoli cells, and (c) an aid in
maintaining the seminiferous epithelium as an immunologically
privileged site (55). What roles the various
(IV) chains have in
these processes remains unknown. In a recent study of mouse testicular
development, it was found that the genes for the
1(IV),
2(IV),
and
5(IV) chains were expressed during embryonic development,
whereas the
3(IV) and
4(IV) chains were expressed postnatally at
a time which coincided with the initiation of spermatogenesis and
expansion of the diameter of the STBM (34, 41, 56). At the protein
level, the
3(IV) chain was found to be incorporated into a
pre-existing STBM composed of the
1(IV) and
2(IV) chains. Whether
the
5(IV) chain pre-existed with the
1(IV) and
2(IV) chains
was not determined. These observations suggested that the
3(IV)
chain is crucial for spermatogonial proliferation. However, recent work
in which the
3(IV) chain was deleted in male mice resulted in
apparently normal fertility (57). Assuming that COL4A5 gene mutations
cause defective assembly of the
3(IV) chain in STBM, as they do in
renal glomerular BM, and that the
3(IV) and
5(IV) chains are
crucial for spermatogenesis, then male Alport patients would be
infertile (58). However, most Alport patients are fertile and only
class I Alport patients lack offspring. Hence, the fertility of Alport
patients raises the question of whether the
3(IV) and
5(IV)
chains are required for spermatogenesis in human beings or whether the
structural relationship of the
3(IV) and
5(IV) chains in STBM
differs from that of glomerular BM.
The detection of 6(IV) chains in larger blood vessels (not
capillaries) and the STBM is consistent with
6(IV) chain expression by smooth muscle cells. Peritubular myoid cells have smooth
cytoskeletal elements including desmin and smooth muscle
-actin (59,
60). Defects in the
6(IV) chain have been associated with
leiomyomatosis, a benign proliferation of smooth muscle cells (7). It
should be interesting to determine if the thickening of the peritubular myoid cell layer which is associated with infertility (61-63) is also
associated with defective or altered
6(IV) chain synthesis.
The technical assistance of Parvin Todd is greatly appreciated.