Seminiferous Tubule Basement Membrane
COMPOSITION AND ORGANIZATION OF TYPE IV COLLAGEN CHAINS, AND THE LINKAGE OF alpha 3(IV) AND alpha 5(IV) CHAINS*

(Received for publication, February 11, 1997, and in revised form, April 28, 1997)

Tesfamichael Z. Kahsai , George C. Enders Dagger , Sripad Gunwar , Charlott Brunmark §, Jörgen Wieslander §, Raghuram Kalluri , Jing Zhou par , Milton E. Noelken and Billy G. Hudson **

From the Departments of Biochemistry and Molecular Biology, and Dagger  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 par  Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

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 alpha -chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of alpha 3(IV) chain (40%) and the alpha 4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six alpha (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 alpha (IV) chains, were characterized for their alpha (IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with alpha (IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the alpha 1(IV) and alpha 2(IV) chains and two novel networks, one composed of the alpha 1(IV)-alpha 6(IV) chains and the other composed of the alpha 3(IV)-alpha 6(IV) chains. The results establish a structural linkage between the alpha 3(IV) and alpha 5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the alpha 5(IV) chain cause defective assembly of the alpha 3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.


INTRODUCTION

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 alpha 1(IV) to alpha 6(IV) (1-8). The entire coding sequences for all six human alpha (IV) chains and certain alpha (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 alpha (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 alpha 1(IV) and alpha 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 alpha (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 [alpha 1(IV)]2alpha 2(IV) and [alpha 3(IV)]2alpha 4(IV) (24, 25). Whether the alpha 5(IV) and alpha 6(IV) chains occur in some combination with alpha 1(IV) to alpha 4(IV) or in separate molecules remains unknown.

The six alpha (IV) chains differ considerably with respect to tissue distribution. At the protein level, immunochemical studies have shown that the alpha 1(IV) and alpha 2(IV) chains have a ubiquitous distribution whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains have a restricted distribution in both human and rodent tissues (26-29). At the mRNA level, the relative expression of the alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains varies greatly among a variety of human tissues, including a variation in the ratio of expression of the alpha 3(IV) and alpha 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 alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains have all been implicated in the pathogenesis of human diseases (1, 7, 30-33). In Goodpasture syndrome, the alpha 3(IV) chain is the target for the pathogenic autoantibodies. In Alport syndrome, the COL4A5 gene encoding the alpha 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 alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 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 alpha (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 alpha (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 alpha 3(IV) chain at the initiation of spermatogenesis (34). The results reveal that STBM contains the highest percentage of the alpha 3(IV) chain of any BM collagen thus far studied and that it exists in a novel supramolecular complex comprised of the alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains.


EXPERIMENTAL PROCEDURES

Materials

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.

Antisera for Use in Immunocytochemistry and Immunoblotting

The antisera used for primary antibody were either from patients with Goodpasture syndrome (autoantiserum) and Alport syndrome (alloantiserum) both directed against alpha 3(IV) NC1 (36, 37) or from rabbits immunized with the monomeric subunits of alpha 1(IV) and alpha 2(IV) NCI domain of type IV collagen (38) or anti-alpha 3(IV) (38), anti-alpha 4(IV) (38), anti-alpha 5(IV) (39), or anti-alpha 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.

Immunocytochemical Localization of alpha (IV) Chains in Bovine Testis

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 alpha 1(IV)/alpha 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 alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 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-alpha 1(IV)/alpha 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.

Preparation of STBM

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 Pseudolysin

STBM (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 Microscopy

The 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 Hexamer

NC1 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 Analysis

One-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 Analysis

Amino-terminal amino acid sequence analysis for alpha 3(IV) NC1 monomer was performed at the University of Kansas Medical Center Biotechnology Support Facility. A lyophilized alpha 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.

Immunoblotting

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 Techniques

Quantitation 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 Chromatography

Two affinity chromatographic columns were prepared using monoclonal antibodies mAb6 and mAb17, specific for alpha 1(IV) NC1 and alpha 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).


RESULTS

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 alpha 1(IV)/alpha 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-alpha l(IV)/alpha 2(IV) antisera, no specific localization was detected (Fig. 1A). In contrast, the alpha 3(IV) chain was localized to the STBM using Alport alloantibody or Goodpasture autoantibody (Fig. 1C) both of which are directed against the alpha 3(IV) chain (3, 36, 37) and using antipeptide antisera to alpha 3(IV) NC1 (Fig. 1E). Similarly, the alpha 4(IV) and alpha 5(IV) chains were localized to STBM (Fig. 1, F and G). The alpha 6(IV) chain was localized to the STBM and larger blood vessel BM (Fig. 1H). Thus, the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are exclusively localized to the STBM and the STBM is comprised of all six alpha (IV) chains.


Fig. 1. Localization of alpha 1/alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains in sections of bovine testis. In all panels of the figure, the large filled arrow points to the STBM, the small filled arrowhead points to interstitial extracellular matrix, and the open arrow points to blood vessels; bar length, 30 µm. Immunoperoxidase localization of type IV collagens chains alpha 1/alpha 2(IV), using rabbit anti-alpha 1/alpha 2(IV) antisera (panel B) with normal rabbit serum (panel A) localized with the (1:1000 dilution). The STBM, extracellular matrixes in the interstitial space, contain alpha 1/alpha 2(IV) chains. Localization of alpha 3(IV) chains using antibodies from an Alport patient (panel C), with control (no primary antibody, panel D), for nonspecific binding of the secondary antibody (1:2000 dilution). Note that most of the immunoreactivity using anti-peptide alpha 3(IV) antisera also appears confined to the STBM (arrows, panel E). Anti-synthetic peptide chain antisera specific for alpha 4(IV) NC1 (panel F) and alpha 5(IV) NC1 (panel G) gave similar restricted localization to the STBM. Anti-synthetic peptide chain antisera specific for alpha 6(IV) NC1 localized to the STBM and larger blood vessel basement membrane (panel H).
[View Larger Version of this Image (122K GIF file)]

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).


Fig. 2. Purification and analysis of NC1 hexamer from STBM. Panel A, unbound material from a DE52 anion-exchange column was separated by gel filtration on Sephacryl S-300. Pool I contains the 7 S material; pool II, NC1 hexamer; pool III, polypeptides which are identified as monomeric and dimeric species of the NC1 domain. Panel B, electron micrograph of NC1 hexamer (pool II, A) of TBM. Panel C, pool II, NC1 hexamers dissociated with 6 M guanidine-HCl and rerun on a Sephacryl S-200 gel filtration column. The resulting profile shows the dissociation of hexamers into dimers and monomers in 6 M guanidine-HCl. Panel D, SDS-PAGE analysis of NC1 hexamer before reduction (lane 2), and after reduction with 2-mercaptoethanol (lane 3). Molecular weight markers are shown in lane 1.
[View Larger Version of this Image (42K GIF file)]

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 alpha 1(IV), alpha 2(IV), and alpha 5(IV) NC1 monomers; fraction II contained alpha 1(IV), alpha 2(IV), and alpha 5(IV) NC1 dimers; fraction III contained alpha 4(IV) NC1 monomers and dimers and alpha 6(IV) NC1 dimers; and fraction IV contained alpha 3(IV) NC1 monomers and dimers and trace amounts of alpha 5(IV) NC1. In each case, dimers have apparent size isoforms, and both monomers and dimers have charge isoforms. The identity of the alpha 3(IV) NC1 monomers, designated alpha 3aNC1 and alpha 3bNC1 in Fig. 4D, was also determined by amino-terminal sequence analysis. The sequence of the first 12 residues of alpha 3aNC1 and alpha 3bNC1 was identical to that previously established for the alpha 3(IV) subunits of bovine glomerular BM (4). Only subunits comprising fraction IV bound Goodpasture autoantibodies and Alport alloantibodies reflecting the presence of alpha 3(IV) NC1 domain as their target as previously established for GBM (3, 36, 37). The relative amount of alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 4(IV) NCl subunits comprising STBM-NCl hexamer was determined from the HPLC profile (Fig. 3B). The relative amount of alpha 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 alpha 3(IV) (40%) and alpha 4(IV) NCl domains (18%) of all mammalian BMs for which quantities have been reported.


Fig. 3. Identification of STBM hexamer subunits by two-dimensional gel electrophoresis and reversed-phase HPLC. Panel A, two-dimensional electrophoretic pattern of STBM-NC1 hexamer showing a complex pattern of monomeric and dimeric components. The designation of alpha (IV) NC1 components was based on their mobility in relation to those established for bovine GBM (44) and on chain-specific antibody reactivity shown in Fig. 4. Arrowheads designate pI markers. Panel B, resolution of TBM hexamer subunits by C18 reversed-phase HPLC as represented by pools I-IV.
[View Larger Version of this Image (44K GIF file)]


Fig. 4. Identification of NC1 subunits comprising of HPLC fractions (Fig. 3, panel B). HPLC fractions I-IV, shown in panel B (Fig. 3), were analyzed by two-dimensional electrophoresis and the gels stained for proteins by silver-staining (panels A-D) and for reactivity with chain-specific antibodies in panels E-L. Fractions I-IV were each reacted with antibodies for each of the six alpha (IV) chains and only those that gave positive reaction are shown. Fractions I and II reacted with anti-[alpha 1/alpha 2](IV) NC1 antibody (panels E and F) and anti-alpha 5(IV) antibody (panels I and J); fraction III reacted with anti-alpha 4(IV) antibody (panel G) and anti-alpha 6(IV) (panel K), fraction IV reacted with anti-alpha 3(IV) antibody (panels D and H) and Goodpasture antibody (panel L). Fraction IV also reacted with Alport alloantibody (data not shown), reflecting the presence of alpha 3(IV) NC1.
[View Larger Version of this Image (84K GIF file)]

Table I. Subunit composition of NC1 hexamers from several basement membranes


Basement membrane Chain identity of NC1 subunits
 alpha 1 + alpha 2  alpha 3 + alpha 4  alpha 5  alpha 3  alpha 4

relative abundance (%)
Seminiferous tubule BMa 41 55 4 38 17
Glomerular BMb 70 27 3 16 11
Alveolar BMc 84 16 NDd 5 11
Lens BMe 94 6 ND 3 3
Placenta BMe 98 2 ND 1 1
EHS matrixf 100 0 ND 0 0

a Values for alpha 1 + alpha 2, alpha 3, and alpha 4 were determined from an HPLC profile for TBM (Fig. 3) and includes both monomer and dimers of NC1 subunits. The value for alpha 5 is a minimum value based on the amount in an affinity chromatography pool D (Fig. 11) that represents 49% of the total NC1.
b Values were taken from Ref. 43 except for alpha 5, which was taken from Ref. 64.
c Values were taken from Ref. 37.
d ND, not determined.
e Values were taken from Ref. 43.
f ESH (Engelbreth-Holm-Swarm tumor matrix); values were taken from Ref. 49.

Organization of Type IV Collagen in STBM

Pseudolysin-soluble STBM

STBM 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 alpha 1(IV) and alpha 2(IV) NC1 monomers and dimers. The HPLC profile (panel A) supports this identity and shows that alpha 1(IV) and alpha 2(IV) NC1 domains comprise >95% of the NC1 domains, reflecting that the triple-helical molecules shown in Fig. 5 are comprised of alpha 1(IV) and alpha 2(IV) chains.


Fig. 5. Electron microscopy of pseudolysin-soluble STBM. Most of the truncated protomers of type IV collagen exist as dimers (NC1:NC1) with a triple-helical length of 280-330 nm and NC1 diameter of 15-17 nm. Bar length, 100 nm.
[View Larger Version of this Image (130K GIF file)]


Fig. 6. Analysis of pseudolysin-soluble and pseudolysin-insoluble STBM by two-dimensional gel electrophoresis and reversed-phase HPLC. Panels A and C are HPLC profiles of NC1 hexamers prepared from pseudolysin-soluble and pseudolysin-insoluble STBM, respectively. The shaded area on the HPLC profile represents the alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains. Panels B and D are two-dimensional electrophoretic patterns of the pseudolysin-soluble and pseudolysin-insoluble STBM, respectively.
[View Larger Version of this Image (61K GIF file)]

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-alpha 1(IV) NC1 and anti-alpha 2(IV) NC1) using a strategy previously described (3, 25). When the NC1 hexamer was applied to an anti-alpha 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 alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 4(IV) NC1 domains and homodimers of alpha 1(IV), alpha 3(IV), and alpha 4(IV) NC1 domains, but no alpha 2(IV) NC1 dimer. Thus, this population(s) of NC1 hexamer is comprised of alpha 3(IV) NC1 in association with alpha 1(IV), alpha 2(IV), and alpha 4(IV) NC1. It represents 2% of the total NC1 hexamer of STBM and it is designated hexamer population A.


Fig. 7. Affinity chromatography of NC1 hexamer from pseudolysin-soluble STBM on an anti-alpha 3(IV) antibody column, and analysis of the bound fraction. The NC1 hexamer from pseudolysin-soluble STBM was resolved into a bound and unbound fraction (panel A) on an anti-alpha 3(IV) antibody column. The bound fraction was analyzed by two-dimensional gel electrophoresis and stained with Coomassie Brilliant Blue R-250 (panel B). R-1 represents a possible heterodimer region of alpha 1(IV) and alpha 3(IV) chains. Immunoblot analysis of the bound fraction with chain-specific antibodies for alpha 1(IV)-alpha 6(IV) NC1 (panels C-G) revealed that it contained alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 4(IV) NC1.
[View Larger Version of this Image (37K GIF file)]

The unbound fraction was then applied to the anti-alpha 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 alpha 1(IV) and alpha 2(IV) NCl (Fig. 8B) in the immunoblot analysis. Furthermore, the bound fraction showed strong reactivity with the antibodies to alpha 1(IV) and alpha 2(IV) NC1 domain (Fig. 8C), but not with the antibodies to alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV)NCl (data not shown). Therefore, this NCl hexamer population, which did not bind to the anti-alpha 3(IV) affinity column but bound to the anti-alpha 1(IV) affinity column, contained only alpha 1(IV) and alpha 2(IV) chains. Thus, this population(s) of NCl hexamer A (Fig. 11) is composed of alpha 1(IV) NC1 associated with an alpha 2(IV) NC1. It represents 14% of the total NC1 hexamer of STBM and is designated hexamer population B.


Fig. 8. Pseudolysin-soluble STBM: affinity chromatography of unbound NC1 hexamer (Fig. 7) on an anti-alpha 1(IV) column and analysis of the bound fraction. All of the unbound NC1 hexamer from the anti-alpha 3(IV) column (Fig. 7A) bound to the anti-alpha 1(IV) antibody column (panel A). The bound fraction was analyzed by two-dimensional gel electrophoresis and staining with Coomassie Brilliant Blue R-250 (panel B). Immunoblot analysis of the bound fraction with chain-specific antibodies for alpha 1(IV)/alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV) NC1, and alpha 6(IV) NC1 domains, respectively, revealed that it contained only alpha 1(IV) and alpha 2(IV) NC1 domains (panel C); the negative blots for alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) are not shown.
[View Larger Version of this Image (38K GIF file)]


Fig. 11. Illustration of NC1 hexamers of STBM. Four distinct populations of NC1 hexamers, designated A to D, were deduced from the studies of the pseudolysin-soluble and pseudolysin-insoluble fraction of STBM. Hexamers were subfractionated using two-affinity columns, an anti-alpha 1(IV) antibody column and an anti-a3(IV) antibody column. In each hexamer population, one subunit designated alpha 1(IV) NC1 or alpha 3(IV) NC1, reflecting on which column the hexamer bound. The relative distribution of the four populations is presented as percentage of total hexamer of STBM. The hexamer populations reflect specific organizations of alpha (IV) chains within triple-helical isoforms that are connected through NC1-NC1 interactions. Population B likely reflects the existence of the classical type IV collagen molecule in STBM having a chain composition of [alpha 1(IV)]2 alpha 2(IV). Populations C and D represent novel organizations of the alpha 1-alpha 6(IV) chains in STBM.
[View Larger Version of this Image (37K GIF file)]

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% alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) NC1 monomers and dimers, and the remainder (<15%) of alpha 1(IV) and alpha 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-alpha 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 alpha 1/alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV)NCl at the monomer and dimer regions (Fig. 9, C-G). Thus, the NC1 hexamer that bound to the anti-alpha 1(IV) affinity column bound NCl hexamer contained all six alpha (IV) chains. It is composed of alpha 1(IV) in association with alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) NC1 domains. It represents 35% of the total NC1 hexamer of STBM and is designated hexamer population C.


Fig. 9. Pseudolysin-insoluble STBM: affinity chromatography of NC1 hexamer (Fig. 7) on an anti-alpha 1(IV) column and analysis of the bound fraction. NC1 hexamer from pseudolysin-insoluble STBM was applied to an anti-alpha 1(IV) antibody column and it resolved into a bound and an unbound fraction (panel A). The bound fraction was analyzed by two-dimensional gel electrophoresis and staining with Coomassie Brilliant Blue R-250 (panel B). R-1, R-2, and R-3 represent the possible regions for alpha 4(IV)-alpha 6(IV), alpha 1(IV)-alpha 3(IV) and alpha 1(IV)-alpha 5(IV), and alpha 1(IV)-alpha 3(IV) NC1 heterodimers, respectively. Immunoblot analysis of the bound fraction with chain-specific antibodies for alpha 1(IV)/alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV) NC1, and alpha 6(IV) NC1 domains, respectively (panels C-G), revealed that it contained alpha 1(IV)/alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) NC1 domains.
[View Larger Version of this Image (42K GIF file)]

When the unbound fraction from the anti-alpha 1(IV) NCl affinity column was applied to the anti-alpha 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 alpha 3(IV) NCl-alpha 6(IV) NCl domains and the absence of alpha 1(IV) and alpha 2(IV) NC1 domains. Thus, this population(s) of NCl hexamer is composed of alpha 3(IV) NC1 domains in association with alpha 4(IV), alpha 5(IV), alpha 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% alpha 3(IV) NC1, 27% alpha 4(IV) NC1 and alpha 6(IV) NC1, and 7% alpha 5(IV) NC1. The alpha 4(IV) content represents the majority of the 27% of alpha 4(IV) plus alpha 6(IV) NC1 domains because alpha 6(IV) NC1 represents only a small fraction, as is shown by the relative abundance of spots (Fig. 4).


Fig. 10. Pseudolysin-insoluble STBM: affinity chromatography of unbound NC1 hexamer (Fig. 9A) on an anti-alpha 3(IV) column and analysis of the bound fraction. Greater than 90% of the unbound fraction from the anti-alpha 1(IV) column (Fig. 9A) bound to an anti-alpha 3(IV) antibody column (panel A). The bound fraction was analyzed by two-dimensional gel electrophoresis and staining with Coomassie Brilliant Blue R-250 (panel B). R-1 and R-2 regions represent the possible regions for alpha 4(IV)-alpha 6(IV) and alpha 3(IV)-alpha 5(IV) heterodimers, respectively. Immunoblot analysis of the bound fraction with chain-specific antibodies for alpha 1(IV)-alpha 6(IV) NC1 (panels C-G) revealed that it contained alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) NC1 domains.
[View Larger Version of this Image (37K GIF file)]


DISCUSSION

STBM was found to be composed of all six alpha -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 alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains are usually regarded as the minor chains of BMs in relation to the abundant alpha 1(IV) and alpha 2(IV) chains. However, in the case of STBM, the alpha 3(IV)-alpha 6(IV) chains collectively represent more than 80% of the chains, with the alpha 3(IV) and alpha 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 alpha (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 alpha 3(IV) and the remaining five chains could be alpha 1(IV), alpha 2(IV), alpha 3(IV), or alpha 4(IV), or a combination of these chains. The existence of an alpha 3(IV) homohexamer cannot be dismissed, but a homohexamer of alpha 1(IV), alpha 2(IV), or alpha 4(IV) can be. Second, in population B, in an individual hexamer, one chain of the two triple-helical isoforms is alpha 1(IV) and the remaining five chains could be alpha 1(IV) or alpha 2(IV) or a combination of alpha 1(IV) and alpha 2(IV) chains. The existence of an alpha 1(IV) homohexamer cannot be dismissed, but a homohexamer of alpha 2(IV) can be. Third, in population C, in an individual hexamer, one chain of the two-triple helical isoforms is alpha 1(IV) and the remaining five chains could be either alpha 1(IV), alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV), or alpha 6(IV) or a combination of these chains. The existence of an alpha 1(IV) homohexamer cannot be dismissed, but a homohexamer of alpha 2(IV), alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 6(IV) can be. Fourth, in population D, in an individual hexamer, one chain of the two triple helical isoforms is alpha 3(IV) and the remaining five chains could be either alpha 3(IV), alpha 4(IV), alpha 5(IV), or alpha 6(IV) or a combination of these chains. The existence of an alpha 3(IV) homohexamer cannot be dismissed, but a homohexamer of alpha 4(IV), alpha 5(IV), and alpha 6(IV) can be. Fifth, the composition of populations A-D clearly reveals that the six alpha (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 alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 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 alpha (IV) chains can assemble randomly, and 18 if they assemble analogously to the well characterized [alpha 1(IV)]2alpha 2(IV) isoform from the EHS matrix (24), wherein an alpha 3(IV) or alpha 5(IV) chain can replace an alpha 1(IV) chain and an alpha 4(IV) or alpha 6(IV) chain can replace alpha 2(IV) chain. In these configurations, the isoforms would have the generic composition: [alpha 1(IV)-like]2alpha 2(IV)-like, relating to their primary structures which are categorized as alpha 1(IV)-like and alpha 2(IV)-like. Clearly, population B is consistent with the [alpha 1(IV)]2alpha 2(IV) isoform. The composition of population D provides the first evidence for an [alpha 3(IV)]2alpha 4(IV) isoform devoid of alpha 5(IV) and alpha 6(IV) chains, as well as an isoform that connects an alpha 3(IV) chain with an alpha 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: [alpha 3(IV)]2alpha 4(IV), alpha 3(IV)alpha 4(IV)alpha 5(IV), alpha 3(IV)alpha 5(IV)alpha 6(IV), and [alpha 5(IV)]2alpha 6(IV). In addition to these putative isoforms and the [alpha 1(IV)]2alpha 2(IV) isoform, the chain content of populations A and C is consistent with the presence of [alpha 1(IV)]2alpha 4(IV), [alpha 1(IV)]2alpha 6(IV), [alpha 3(IV)]2alpha 6(IV), alpha 1(IV)alpha 2(IV)alpha 3(IV), alpha 1(IV)alpha 2(IV)alpha 5(IV), alpha 1(IV)alpha 3(IV)alpha 4(IV), and alpha 1(IV)alpha 3(IV)alpha 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: [alpha 1(IV)]2alpha 2(IV), [alpha 3(IV)]2alpha 4(IV), alpha 3(IV)alpha 4(IV)alpha 5(IV), and alpha 3(IV)alpha 5(IV)alpha 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.


Fig. 12. Hypothetical STBM collagen IV NC1-linked isoforms and their association products. Panel A, evidence consistent with an NC1-linked dimer of the [alpha 1(IV)]2alpha 2(IV) isoform (open circle , unfilled) was obtained by electron microscopy (Fig. 5) of the collagen IV truncated isoform excised by pseudolysin at 4 °C, and analysis of the NC1 hexamers derived from it (Fig. 11, populations A and B). Evidence consistent with an [alpha 3(IV)]2alpha 4(IV) isoform (open circle , black filling) and an alpha 3(IV)alpha 5(IV)[alpha 4(IV) or alpha 6(IV)] isoform (&cjs0383;, black and white striped filling) was obtained by analysis of NC1 hexamer populations C and D (Fig. 11). Evidence consistent with the lower three NC1-linked isoforms in panel A was obtained by analysis of NC1 hexamer populations C and D (Fig. 11). In panels B, C, and D are hypothetical collagen IV octamers whose structures are consistent with the analysis of NC1 hexamer populations B, C, and D (Fig. 11), respectively. The octamers could occur either in separate networks or as part of a single network. It should be noted that the chain composition of the isoforms, and thus the isoform composition of the octamers, is not completely established.
[View Larger Version of this Image (25K GIF file)]

NC1 hexamer population B reflects the classical organization in which the alpha 1(IV) chain is linked to the alpha 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 alpha 1(IV) and alpha 2(IV) chains can form an independent network in tissues that also contain the alpha 3(IV)-alpha 6(IV) chains. Immunolocalization studies of the kidney show that only alpha 1(IV) and alpha 2(IV) chains are found in the mesangium and artery, whereas chains alpha 1(IV) through alpha 5(IV) are found in glomerular and tubular BM (26-28). Thus, isoforms containing alpha 1(IV) and alpha 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 alpha 1(IV)/alpha 2(IV) network is that these chains appear to have a ubiquitous occurrence in tissues, in contrast to the restricted distribution of the other alpha (IV) chains (1). In STBM, hexamer population B contains NC1 domains derived only from alpha 1(IV) and alpha 2(IV) chains. These results are consistent with an independent network that is composed of alpha 1(IV) and alpha 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 alpha 1(IV) chain is linked to alpha 2(IV)-alpha 6(IV) chains, suggesting a network that is composed of all six alpha (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 alpha (IV) chains exist in STBM. The possibility of this complicated type of network had been realized when the alpha 3(IV) and alpha 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 alpha 3(IV) chain is linked to the alpha 4(IV), alpha 5(IV), and alpha 6(IV) chains. At one extreme, the alpha 3(IV) chain could be linked to five alpha 4(IV) chains in the hexamer, forming two triple-helical isoforms that are connected through their respective NC1 domains, or to five alpha 5(IV) chains or five alpha 6(IV) chains. On the other extreme, the alpha 3(IV) chain could be linked to five chains in the hexamer in which the alpha 4(IV), alpha 5(IV), and alpha 6(IV) chain are components. In either extreme, population D links an alpha 3(IV) chain with an alpha 5(IV) chain. The results are consistent with a separate network composed of only alpha 3(IV), alpha 4(IV), alpha 5(IV), and alpha 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 alpha (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 alpha 3(IV) chain and an alpha 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 alpha 5(IV) chain, cause defective assembly of the alpha 3(IV) chain in the glomerular BM (1, 29, 36, 52, 53). A structural linkage between the alpha 3(IV) and alpha 5(IV) chains suggests that the alpha 5(IV) chain may be required for the incorporation of the alpha 3(IV) chain into a triple-helical isoform containing both alpha 3(IV) and alpha 5(IV) chains or into two triple-helical isoforms, connected through NC1 interactions, in which the alpha 3(IV) chain is contained within one isoform and the alpha 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 alpha 1(I) chain cause defective incorporation of the alpha 2(I) chain (54).

The structural linkage between the alpha 3(IV) and alpha 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 alpha (IV) chains have in these processes remains unknown. In a recent study of mouse testicular development, it was found that the genes for the alpha 1(IV), alpha 2(IV), and alpha 5(IV) chains were expressed during embryonic development, whereas the alpha 3(IV) and alpha 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 alpha 3(IV) chain was found to be incorporated into a pre-existing STBM composed of the alpha 1(IV) and alpha 2(IV) chains. Whether the alpha 5(IV) chain pre-existed with the alpha 1(IV) and alpha 2(IV) chains was not determined. These observations suggested that the alpha 3(IV) chain is crucial for spermatogonial proliferation. However, recent work in which the alpha 3(IV) chain was deleted in male mice resulted in apparently normal fertility (57). Assuming that COL4A5 gene mutations cause defective assembly of the alpha 3(IV) chain in STBM, as they do in renal glomerular BM, and that the alpha 3(IV) and alpha 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 alpha 3(IV) and alpha 5(IV) chains are required for spermatogenesis in human beings or whether the structural relationship of the alpha 3(IV) and alpha 5(IV) chains in STBM differs from that of glomerular BM.

The detection of alpha 6(IV) chains in larger blood vessels (not capillaries) and the STBM is consistent with alpha 6(IV) chain expression by smooth muscle cells. Peritubular myoid cells have smooth cytoskeletal elements including desmin and smooth muscle alpha -actin (59, 60). Defects in the alpha 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 alpha 6(IV) chain synthesis.


FOOTNOTES

*   The work was supported in part by National Institutes of Health Grant DK 18381 (to B. G. H.) and University of Kansas Medical Center Grant BRSG SO7 RR05373 (to G. C. E.).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 Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421.
1   The abbreviations used are: BM, basement membrane; NC1, noncollagenous domain; STBM, seminiferous tubule basement membrane, i.e. extracellular matrix material of the seminiferous tubule lamina propria; alpha 1(IV)/alpha 2(IV), alpha 1(IV) and/or alpha 2(IV); HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
2   Pseudolysin (EC 3.4.24.26), from P. aeruginosa, was originally called Pseudomonas elastase, the name we used in previous reports.
3   The NC1 domain is excised from the BM suprastructure by collagenase digestion isolated as hexamer. The NC1 hexamer is composed of subunits that correspond to the NC1 domain of the six alpha  chains comprising two adjoining isoforms (triple-helical molecules). The kind of stoichiometry of subunits in a hexamer depends on the chain composition of the isoform and the kind of isoforms associated through NC1-NC1 interactions (1).
4   In population D, alpha 5(IV) NC1 monomers and dimers are 7% by weight of the total NC1 content. A value of 16.6% would be required for all triple helical molecules to contain an alpha 5(IV) chain. Thus, the high abundance of the alpha 3(IV) NC1 (66%) and alpha 4(IV) NC1 (27%) domains reflects a fraction of the triple helical molecules containing exclusively alpha 3(IV) and alpha 4(IV) chains with a presumed composition of [alpha 3(IV)]2alpha 4(IV).

ACKNOWLEDGEMENT

The technical assistance of Parvin Todd is greatly appreciated.


REFERENCES

  1. Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036 [Free Full Text]
  2. Butkowski, R. J., Langeveld, J. P. M., Wieslander, J., Hamilton, J., and Hudson, B. G. (1987) J. Biol. Chem. 262, 7874-7877 [Abstract/Free Full Text]
  3. Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380 [Abstract/Free Full Text]
  4. Gunwar, S., Saus, J., Noelken, M. E., and Hudson, B. G. (1990) J. Biol. Chem. 265, 5466-5469 [Abstract/Free Full Text]
  5. Hostikka, S. L., Eddy, R. L., Hoyhtya, M., Shows, T. B., and Tryggvason, K. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1606-1610 [Abstract]
  6. Pihlajaniemi, T., Pohjolainen, E.-R., and Myers, J. C. (1990) J. Biol. Chem. 265, 13758-13766 [Abstract/Free Full Text]
  7. Zhou, J., Mochizuki, T., Smeets, H., Antignac, C., Laurila, P., de Paepe, A., Tryggvason, K., and Reeders, S. T. (1993) Science 261, 1167-1169 [Medline] [Order article via Infotrieve]
  8. Soininen, R., Haka-Risku, T., Prockop, D. J., and Tryggvason, K. (1987) FEBS Lett. 225, 188-194 [CrossRef][Medline] [Order article via Infotrieve]
  9. Hostikka, S. L., and Tryggvason, K. (1988) J. Biol. Chem. 263, 19488-19493 [Abstract/Free Full Text]
  10. Zhou, J., Hertz, J. M., Leinonen, A., and Tryggvason, K. (1992) J. Biol. Chem. 267, 12475-12481 [Abstract/Free Full Text]
  11. Morrison, K. E., Mariyama, M., Yang-Feng, T. L., and Reeders, S. T. (1991) Am. J. Hum. Genet. 49, 545-554 [Medline] [Order article via Infotrieve]
  12. Mariyama, M., Leinonen, A., Mochizuki, T., Tryggvason, K., and Reeders, S. T. (1994) J Biol. Chem. 269, 23013-23017 [Abstract/Free Full Text]
  13. Sugimoto, M., Oohashi, T., Yoshioka, H., Matsuo, N., and Ninomiya, Y. (1993) FEBS Lett. 330, 122-128 [CrossRef][Medline] [Order article via Infotrieve]
  14. Muthukumaran, G., Blumberg, B., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6310-6317 [Abstract/Free Full Text]
  15. Oohashi, T., Sugimoto, M., Mattei, M.-G., and Ninomiya, Y. (1994) J. Biol. Chem. 269, 7520-7526 [Abstract/Free Full Text]
  16. Zhou, J., Ding, M., Zhao, Z., and Reeders, S. T. (1994) J. Biol. Chem. 269, 13193-13199 [Abstract/Free Full Text]
  17. Leinonen, A., Mariyama, M., Mochizuki, T., Tryggvason, K., and Reeders, S. T. (1994) J. Biol. Chem. 269, 26172-26177 [Abstract/Free Full Text]
  18. Saus, J., Quinones, S., MacKrell, A., Blumberg, B., Muthukumaran, G., Pihlajaniemi, T., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6318-6324 [Abstract/Free Full Text]
  19. Morrison, K. E., Germino, G. G., and Reeders, S. T. (1991) J. Biol. Chem. 266, 34-39 [Abstract/Free Full Text]
  20. Turner, N., Mason, P. J., Brown, R., Fox, M., Povey, S., Rees, A., and Pusey, C. D. (1992) J. Clin. Invest. 89, 592-601 [Medline] [Order article via Infotrieve]
  21. Quinones, S., Bernal, D., Garcia-Sogo, M., Elena, S. F., and Saus, J. (1992) J. Biol. Chem. 267, 19780-19784 [Abstract/Free Full Text]
  22. Mariyama, M., Kalluri, R., Hudson, B. G., and Reeders, S. T. (1992) J. Biol. Chem. 267, 1253-1258 [Abstract/Free Full Text]
  23. Long, G. C., Thomas, M., and Brodsky, B. (1995) Biopolymers 35, 621-628 [Medline] [Order article via Infotrieve]
  24. Brazel, D., Oberbäumer, I., Dieringer, H., Babel, W., Glanville, R. W., Deutzmann, R., and Kühn, K. (1987) Eur. J. Biochem. 168, 529-536 [Abstract]
  25. Johansson, C., Butkowski, R., and Wieslander, J. (1992) J. Biol. Chem. 267, 24533-24537 [Abstract/Free Full Text]
  26. Ninomiya, Y., Kagawa, M., Iyama, K.-I., Naito, I., Kishiro, Y., Seyer, J. M., Sugimoto, M., Oohashi, T., and Sado, Y. (1995) J. Cell Biol. 130, 1219-1229 [Abstract]
  27. Miner, J. H., and Sanes, J. R. (1994) J. Cell Biol. 127, 879-891 [Abstract]
  28. Butkowski, R. J., Wieslander, J., Kleppel, M., Michael, A. F., and Fish, A. J. (1989) Kidney Int. 35, 1195-1202 [Medline] [Order article via Infotrieve]
  29. Peissel, B., Geng, L., Kalluri, R., Kashtan, C., Rennke, H. G., Gallo, G. R., Yoshioka, K., Sun, M. J., Hudson, B. G., Neilson, E. G., and Zhou, J. (1995) J. Clin. Invest. 96, 1948-1957 [Medline] [Order article via Infotrieve]
  30. Barker, D. F., Hostikka, S. L., Zhou, J., Chow, L. T., Oliphant, A. R., Gerken, S. C., Gregory, M. C., Skolnick, M. H., Atkin, C. L., and Tryggvason, K. (1990) Science 248, 1224-1227 [Medline] [Order article via Infotrieve]
  31. Tryggvason, K., Zhou, J., Hostikka, S. L., and Shows, T. B. (1993) Kidney Int. 43, 38-44 [Medline] [Order article via Infotrieve]
  32. Lemmink, H. H., Mochizuki, T., van der Heuvel, L. P., Schroder, C. H., Barrientos, A., Monnens, L. A., van Oost, B. A., Brunner, H. G., Reeders, S. T., and Smeets, H. J. (1994) Hum. Mol. Genet. 3, 1269-1273 [Abstract]
  33. Mochizuki, T., Lemmink, H. H., Mariyama, M., Antignac, C., Gubler, M. C., Pirson, Y., Verellen-Domoulin, C., Chan, B., Schroder, C. H., Smeets, H. J., and Reeders, S. T. (1994) Nat. Genet. 8, 77-81 [Medline] [Order article via Infotrieve]
  34. Enders, G. C., Kahsai, T. Z., Lian, G., Funabiki, K., Killen, P. D., and Hudson, B. G. (1995) Biol. Reprod. 53, 1489-1499 [Abstract]
  35. Morihara, K. (1995) Methods Enzymol. 248, 242-253 [Medline] [Order article via Infotrieve]
  36. Hudson, B. G., Kalluri, R., Gunwar, S., Weber, M., Ballester, F., Hudson, J. K., Noelken, M. E., Sarras, M., Richardson, W. R., Saus, J., Abrahamson, D. R., Glick, A. D., Haralson, M. A., Helderman, J. H., Stone, W. J., and Jacobson, H. R. (1992) Kidney Int. 42, 179-187 [Medline] [Order article via Infotrieve]
  37. Gunwar, S., Bejarano, P. A., Kalluri, R., Langeveld, J. P. M., Wisdom, B. J., Jr., Noelken, M. E., and Hudson, B. G. (1991) Am. J. Resp. Cell Mol. Biol. 5, 107-112 [Medline] [Order article via Infotrieve]
  38. Gunwar, S., Noelken, M. E., and Hudson, B. G. (1991) J. Biol. Chem. 266, 14088-14094 [Abstract/Free Full Text]
  39. Neilson, E. G., Kalluri, R., Sun, M. J., Gunwar, S., Danoff, T., Mariyama, M., Myers, J. C., Reeders, S. T., and Hudson, B. G. (1993) J. Biol. Chem. 268, 8402-8405 [Abstract/Free Full Text]
  40. Johnson, D. A., Gautsch, J. W., Sportsman, J. R., and Elder, J. H. (1984) Gene Anal. Tech 1, 3-8 [CrossRef]
  41. Lian, G., Miller, K. A., and Enders, G. C. (1992) Biol. Reprod. 47, 316-325 [Abstract]
  42. Enders, G. C., Henson, J. H., and Millette, C. F. (1986) J. Cell Biol. 103, 1109-1119 [Abstract]
  43. Langeveld, J. P. M., Wieslander, J., Timoneda, J., McKinney, P., Butkowski, R. J., Wisdom, B. J., Jr., and Hudson, B. G. (1988) J. Biol. Chem. 263, 10481-10488 [Abstract/Free Full Text]
  44. Wieslander, J., Langeveld, J., Butkowski, R., Jodlowski, M., Noelken, M., and Hudson, B. G. (1985) J. Biol. Chem. 260, 8564-8570 [Abstract/Free Full Text]
  45. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  46. O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021 [Abstract]
  47. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310 [Medline] [Order article via Infotrieve]
  48. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203 [Medline] [Order article via Infotrieve]
  49. Wisdom, B. J., Jr., Gunwar, S., Hudson, M. D., Noelken, M. E., and Hudson, B. G. (1992) Connect. Tissue Res. 27, 225-234 [Medline] [Order article via Infotrieve]
  50. Yurchenco, P. D., and O'Rear, J. J. (1994) Methods Enzymol. 245, 489-518 [Medline] [Order article via Infotrieve]
  51. Hudson, B. G., Wieslander, J., Wisdom, B. J., Jr., and Noelken, M. E. (1989) Lab. Invest. 61, 256-269 [Medline] [Order article via Infotrieve]
  52. Kalluri, R., Weber, M., Netzer, K.-O., Sun, M. J., Neilson, E. G., and Hudson, B. G. (1994) Kidney Int. 45, 721-726 [Medline] [Order article via Infotrieve]
  53. Kalluri, R., van den Heuvel, L. P., Smeets, H. J. M., Schroder, C. H., Lemmink, H. H., Boutaud, A., Neilson, E. G., and Hudson, B. G. (1995) Kidney Int. 47, 1199-1204 [Medline] [Order article via Infotrieve]
  54. Prockop, D. J. (1990) J. Biol. Chem. 265, 15349-15352 [Free Full Text]
  55. Tung, K. (1988) Hosp. Pract. Off. Ed. 23, 191-197 [Medline] [Order article via Infotrieve] , 201-202, 205-206
  56. Vergouwen, R. P. F. A., Jacobs, S. G. P. M., Huiskamp, R., Davids, J. A. G., and de Rooij, D. J. (1991) J. Reprod. Fert. 93, 233-243 [Abstract]
  57. Miner, J. H., and Sanes, J. R. (1996) J. Cell Biol. 135, 1403-1413 [Abstract]
  58. Atkin, C. L., Gregory, M. C., and Border, W. A. (1988) in Diseases of the Kidney (Schrier, R. W., and Gottschalk, C. W., eds), 4th Ed., pp. 617-643, Little, Brown and Co., Boston
  59. Schlatt, S., Weinbauer, G. F., Arslan, M., and Nieschlag, E. (1993) J. Androl. 14, 340-350 [Abstract]
  60. Paranko, J., and Pelliniemi, L. J. (1992) Cell Tissue Res. 268, 521-530 [Medline] [Order article via Infotrieve]
  61. de Krester, D. M., Kerr, J. B., and Paulsen, C. A. (1975) Biol. Reprod. 12, 317-324 [Medline] [Order article via Infotrieve]
  62. Salomon, F., and Hedinger, C. E. (1982) Lab. Invest. 47, 543-554 [Medline] [Order article via Infotrieve]
  63. Davidoff, M. S., Breucker, H., Holstein, A. F., and Seidl, K. (1990) Cell Tissue Res. 262, 253-261 [Medline] [Order article via Infotrieve]
  64. Gunwar, S., Ballester, F., Kalluri, R., Timoneda, J., Chonko, A. M., Edwards, S. J., Noelken, M. E., and Hudson, B. G. (1991) J. Biol. Chem. 266, 15318-15324 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.