Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M312694200 on March 10, 2004

J. Biol. Chem., Vol. 279, Issue 22, 23486-23494, May 28, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/22/23486    most recent
M312694200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hummel, S.
Right arrow Articles by Schneider, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hummel, S.
Right arrow Articles by Schneider, W. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Extracellular Matrices of the Avian Ovarian Follicle

MOLECULAR CHARACTERIZATION OF CHICKEN PERLECAN*

Susanna Hummel{ddagger}, Andreas Osanger{ddagger}, Tarek M. Bajari{ddagger}, Manimalha Balasubramani§, Willi Halfter§, Johannes Nimpf{ddagger}, and Wolfgang J. Schneider{ddagger}

From the {ddagger}Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Institute of Medical Biochemistry, Department of Molecular Genetics, Medical University of Vienna, A-1030 Vienna, Austria and the §Department of Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, November 20, 2003 , and in revised form, March 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In egg-laying species, such as the chicken, the mode of transport of lipoprotein particles from the capillary plasma to endocytic receptors on the oocyte surface is largely unknown. Here we show by molecular characterization that the large prominent heparan sulfate proteoglycan of extracellular matrices, termed perlecan or HSPG2 (the product of the hspg2 gene), is a component of ovarian follicles that may participate in this process. However, although normally a major HSPG of basement membranes or basal laminae, in chicken follicles, perlecan is absent from the membranous structure between the theca interna and granulosa cell layers, which to date has been considered a bona fide basement membrane. Rather, the protein is localized in the extracellular matrix of theca externa cells, which produce this HSPG. Furthermore, in chicken testes, perlecan is localized in the peritubular spaces but in less organized fashion than the classical basement membrane components, agrin and laminin. All five domains and structural hallmarks of chicken perlecan (4071 residues) have been conserved in its mammalian counterparts. We have produced the recombinant domain II (containing low density lipoprotein (LDL) receptor-like binding repeats) of chicken perlecan and demonstrate its capacity to bind LDL and very low density lipoprotein (VLDL), apolipoprotein B-containing lipoproteins ultimately destined for uptake into oocytes via members of the low density lipoprotein receptor family. Binding to perlecan heparan sulfate side chains may facilitate the interaction of lipoproteins with domain II. Based on the current results and on domain-domain interactions revealed by recent ultrastructural investigations of the LDL receptor, nidogen, and laminin (Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., and Deisenhofer, J. (2002) Science 298, 2353-2358 and Takagi, J., Yang, Y., Liu, J. H., Wang, J. H., and Springer, T. A. (2003) Nature 424, 969-974), we propose a novel role of perlecan in mediating plasma-to-oocyte surface transport of VLDL particles.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During the development of the cellular architecture of ovarian follicles in the laying hen, the formation of extracellular matrices and laminar structures by differentiating cells are important processes. The regulation of their synthesis and assembly is one of the prerequisites for the normal growth of the central giant germ cell of the follicles, the oocyte. In the final 7-day period of rapid growth, the oocyte is surrounded by a concentric arrangement of, from the periphery inwards, (i) vascularized connective tissue representing an extension of the follicle stalk; (ii) the theca proper, which can be divided into the theca externa, a broad layer of stratified cells with embedded capillaries, and the theca interna, a narrower zone of loosely packed cells also containing capillaries; (iii) an acellular layer commonly designated as "basal lamina" (iv) an epitheloid layer of granulosa cells; and (v) the zona pellucida. Previous studies (1, 2) have demonstrated that oocyte growth and development are achieved through the unprecedentedly efficient uptake of hepatically synthesized yolk precursor proteins by multifunctional oocytic plasma membrane receptors belonging to the low density lipoprotein receptor (LR)1 gene family. We have previously shown that at least two such receptors, termed LR8 (3-5) and LRP380 (6), mediate yolk formation via uptake from the serum compartment of macromolecules including very low density lipoprotein (VLDL), vitellogenin, and {alpha}2-macroglobulin.

The size of serum-borne VLDL particles, on the order of 40-nm diameter (7, 8), poses a question. How do these large complex molecules gain access to the oocytic plasma membrane in the core of the follicle? Detailed ultrastructural studies in the late 1970s (9) established that the endothelium of capillaries within the theca layer shows extensive fenestrae with widths of up to 50 nm, many of which appear to provide direct access from the lumen to the pericapillary space. Thus, when macromolecules are released from the capillary serum compartment, they first encounter extracellular matrices, from where they must be free to diffuse across the basal lamina, through gaps between granulosa cells, and past the zona pellucida, to home in on high affinity endocytic receptors on the oocyte surface. The so-called basal lamina or basement membrane, which separates granulosa cells from theca interna, has previously been shown to be permeable to serum-derived VLDL particles (10, 11). These studies suggested that the high concentration of the particles residing within the membrane is related to its permeability for VLDL in the fluid phase (11).

Inasmuch as previous studies have not addressed the molecular nature of this follicular membrane to any extent, and as it is generally accepted that the heparan sulfate proteoglycan, perlecan, is an important functional component of basement membranes, we have initiated studies on chicken perlecan. Mammalian perlecans (12, 13) are comprised of five domains, of which only the first, a heparan sulfate-containing region, is unique. The other four domains, from the N terminus, are homologous to the LDL receptor, the N-terminal region of laminin A and B short arms, N-CAM, and the globular C terminus of the laminin A chain, respectively. Molecular characterization of the first avian perlecan shows that this protein has been well conserved in mammalian species and is synthesized by theca cells but is absent from the membrane that separates the theca from the granulosa cell layer. Based on ligand binding results with the recombinant LDL receptor-homologous domain of perlecan, we propose that it may function in extracellular transient retention of yolk precursor molecules released from the capillary bed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Animals—Derco Brown laying hens and roosters (30-40 weeks old) were purchased from Heindl (Vienna, Austria) and maintained on layer's mash with free access to water and feed under a daily light period of 14 h.

Isolation of Chicken Perlecan cDNA—The mouse HSPG2 cDNA fragments BG5 (2.0 kb) and BG7 (2.2 kb) (14) (kindly provided by Dr. J. Hassell, Shriners Hospital for Children, Tampa, FL) were used as probes for screening an oligo(dt)- and random-primed chicken eye {lambda}gt11 cDNA library (Clontech). cDNA probes, derived from the first chicken HSPG2 clone confirmed by sequencing and from subsequently isolated clones, were used to screen further cDNA libraries to obtain full-length chicken HSPG2 cDNA: a chicken muscle {lambda}gt11 cDNA library, oligo(dt)and random-primed (Clontech); a chicken embryo {lambda}gt11 cDNA library, oligo(dt)-primed (Clontech); a chicken follicle {lambda}gt11 cDNA library, oligo(dt)- and random-primed (Bujo et al. (5)); and a laying hen liver ZAP II cDNA library, oligo(dt)- and random-primed (Takara). Inserts were cloned and sequenced. The 5'-end of the coding sequence was obtained by using the 5'-/3'-rapid amplification of cDNA ends Kit (Roche Applied Science) and poly(A)-mRNA isolated from the theca layer with the primer 5'-CACGCTGCCCTCCAGCTCCTTGAT-3', corresponding to nucleotides 412-435; for the first PCR reaction, the primer 5'-GCCATCTGCAGACGCTTCATCTGC-3' corresponding to nucleotides 172-195, was used, and for the second PCR reaction 5'-CGTGTCCTCAGGGAAGGAGCTCTC-3', corresponding to nucleotides 76-99, was used. Further cloning details are available upon request.

RNA Preparation and Northern Blot Analysis—Total RNA was isolated from various chicken tissues using TRI reagent (Molecular Research Center, Inc.). Total RNA (30 µg) was subjected to electrophoresis on a 1% agarose gel in the presence of glyoxal (15), blotted, and covalently bound by UV cross-linking onto a positively charged nylon membrane (Amersham Biosciences). The following probes were used: for chicken perlecan, a 339-bp fragment corresponding to nucleotides 6872-7210; for chicken agrin, the cDNA-clone pBG5 (16); for chicken laminin B1, 845 bp corresponding to nucleotides 363-1207; and for chicken {beta}-actin, 340 bp corresponding to nucleotides 627-966, respectively. The probes were labeled with dCTP32 by random priming, and hybridization was performed under standard conditions. The blots were subsequently exposed to x-ray film with an intensifying screen at -80 °C.

Cloning of the Chicken Perlecan LA Repeats 2-4 into the pMAL Vector—PCR of the chicken HSPG2 ligand-binding domain repeats 2-4, designated perl2-4, was performed using the forward primer 5'-GCGGATCCGCCCCACGGCTGCCGCC-3' with a BamHI site (underlined) corresponding to nucleotides 805-823 and the reverse primer 5'-AGCCCGGGCGGCGGCATGCAGCCGAC-3' with an XmaI site (underlined) corresponding to nucleotides 1165-1183. The PCR product (392 bp) was cloned into the pMAL vector (New England Biolabs), which had been modified to provide a His tag at the C-terminal end of the resulting fusion protein (a kind gift of Dr. D. Blaas in our Department). The construct pMAL-perl-LA2-4 was confirmed by sequencing.

Expression of pMAL-perl2-4, GST·RAP, GST·RAP-Myc, and RAPMyc, Purification and Refolding—The vectors pMAL-perl2-4, pGEXRAP (receptor-associated protein), and pET-15b-MycRAP (17) were expressed in the Escherichia coli strain Top10F' (Invitrogen). After induction with 2 mM isopropyl-1-thio-{beta}-D-galactopyranoside for 4 h, the proteins were purified and folded essentially as described (18). GST·RAP-Myc was purified using glutathione-Sepharose 4B (Amersham Biosciences). As described previously (18), the pMAL-perl2-4 was refolded by dialysis in the presence of RAP-Sepharose. The bound and refolded protein was then eluted from the RAP-Sepharose with 0.5 x TBS + 2 mM CaCl2 (TBS-C), 1 M NH4OH. The eluted proteins were immediately adjusted to pH 8 with 0.1 M HCl and dialyzed against TBS-C for 24 h.

Preparation of Lipoproteins and Cell Uptake Studies—VLDL and LDL were isolated from the serum of laying hens by differential floatation as described previously (19). 125I-VLDL was generated by the iodine monochloride method as described (18) and extensively dialyzed against 50 mM NaCl, 0.2 mM EDTA (pH 7.4) before use and stored at 4 °C. Indicated concentrations represent the protein content. Mouse NIH3T3 cells were grown overnight on culture slides in medium supplemented with lipoprotein-deficient serum in the presence of 1 µM NK104 to maximally induce LDL receptor expression (18) and then incubated for 1 h at 37 °C with LDL alone or in the presence of perl2-4 at the indicated concentrations. Following incubation, the cells were fixed and permeabilized with methanol/acetone (1/1, by volume) at -20 °C, incubated with rabbit anti-chicken apoB antiserum (1:500) (20) followed by 5 µg/ml Alexa Fluor 568-labeled anti-rabbit IgG (Molecular Probes).

Electrophoresis, Transfer to Nitrocellulose, and Ligand and Western Blotting—Proteins were separated by one-dimensional 10% SDS-PAGE under reducing or non-reducing conditions according to Laemmli (21). Molecular weights were estimated with a broad range molecular mass standard (6.5-200 kDa) from Bio-Rad. After electrophoresis, proteins were either visualized with Coomassie Brilliant Blue or electrophoretically transferred to WESTRAN clear signal PVDF membranes (Schleicher & Schuell) in 20 mM Tris-HCl, 0.15 M glycine, 20% methanol, pH 8.4, for 60 min at 14 V on a semidry trans-blot SD transfer cell (Bio-Rad). After transfer, proteins were visualized by staining the membrane with Ponceau S (Sigma) and destaining with H2O. For Western blot analyses, the membranes were blocked with 5% non-fat dry milk in TBS containing 0.1% Tween 20, and incubated with anti-maltose-binding protein (MBP) antiserum (New England BioLabs; 1:1000), anti-perl2-4 antiserum (1:1000), or anti-perl2-4 preimmune serum (1:1000). Bound antibodies were detected with horseradish peroxidase-labeled goat anti-rabbit IgG (1:10,000; Sigma) and an enhanced chemiluminescence (ECL) system (Pierce). For ligand blotting with receptor-associated protein (RAP), blocking was with 5% bovine serum albumin (fatty acid-free, Sigma) in TBS-C. Following incubation with the indicated concentrations of RAP-Myc and GST·RAP, bound RAP-Myc was detected with monoclonal anti-Myc antibodies (1:500), horseradish peroxidase-goat-anti-mouse IgG (1:40,000; Sigma), and an ECL system. For ligand blotting experiments using 125I-VLDL, PVDF membranes were blocked and incubated with 1.5 x 106 cpm/ml 125I-VLDL (specific activity, 530 cpm/ng; protein concentration 1.5 µg/ml) as described previously (19). For competition experiments, 125I-VLDL was coincubated with the indicated concentrations of EDTA, GST·RAP, or unlabeled VLDL. Bound 125I-VLDL was detected by exposing the membrane to x-ray film with intensifying screens at -80 °C.

Immunohistochemical Studies—For immunohistochemistry, the follicles (5-7 mm diameter) and rooster testes were embedded in freezing agent (Microm Austria) and immediately frozen. Cryostat sections of 15 µm were prepared and fixed on SuperFrostR Plus slides (Menzel) with aceton/methanol (1:1) for 10 min. The slides were blocked with 3% inactivated goat serum, 1% milk powder in phosphate-buffered saline (pH 7.4) for 1 h at room temperature and then incubated with different antibodies and antisera for 1 h. After three rinses with phosphate-buffered saline, they were incubated with 1:500 fluorescein isothiocyanate-labeled goat anti-rabbit antibodies or goat anti-mouse antibodies (both from Molecular Probes) for 1 h. After three rinses with phosphate-buffered saline, the specimens were mounted in fluorescent mounting medium (DAKO) and photographed with a fluorescence microscope (Zeiss, Axiovert 135). Counterstaining of cellular nuclei was performed with DAPI. Where indicated, heparinase III (heparitinase; Sigma) treatment of tissue sections was performed in 20 mM Tris-HCl, 4 mM CaCl2, 0.01% bovine serum albumin, and 0.5 units of heparinase III for 3 h at 37 °C.

Antibodies and Antisera—Polyclonal antibodies directed against the isolated major component (termed ggBM1) of the membrane separating the granulosa from theca cells, or against the bacterially expressed perl2-4, were raised in adult female New Zealand White rabbits (4) and are designated anti-ggBM1 or anti-perl2-4. Rabbit antiserum against MBP was obtained from New England Biolabs. For immunohistochemistry, the following additional rabbit polyclonal antisera were used: against chicken agrin (22); against mouse perlecan (kindly provided by the late Dr. R. Timpl, MPI Martinsried, Germany); against chicken fibronectin (Chemicon); furthermore, anti-perl2-4 and anti-ggBM1 antiserum as described above. The following monoclonal antibodies were used: mAb 3H11 against embryonic chicken laminin (22); mAb COL I against collagen I (Sigma); mAb 2E9 against Syndecan-1/Syndecan-3; mAb 3G10 against heparitinase-treated heparan sulfate chains (kindly provided by Dr. G. David, Center of Human Genetics, University of Leuven, Leuven, Belgium); and mAb 5C9 against chick perlecan.2


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Chicken HSPG2—The contiguous 12,216-bp coding sequence of chicken HSPG2 was obtained from aligning multiple overlapping clones obtained by screening several cDNA libraries. In a screen of the oligo(dt)- and random-primed chicken eye {lambda}gt11 cDNA library with two murine HSPG2 cDNAs (14), we obtained seven overlapping cDNA clones ranging in size from 260 to 6300 bp that covered the central 2/3 of the expected full-length sequence. To obtain clones covering the 5'-end as well as the 3'-end of chicken HSPG2, oligo(dt)- and/or random primed chicken muscle, ovarian follicle, and liver {lambda}gt11 cDNA libraries were screened with flanking clones, and 5'/3'rapid amplification of cDNA ends-PCR cloning was performed. A total of 18 overlapping clones for chicken HSPG2 were sequenced on both strands and span a total of 12,255 bp. They encoded one contiguous open reading frame plus 39 nucleotides of 3'-untranslated sequence. The nucleotide and the predicted amino acid sequence have been submitted to the EMBL Nucleotide Sequence Database (accession number AJ584653 [GenBank] ). The open reading frame of 12,216 bp encodes 4071 amino acid residues with a calculated molecular size of 432 kDa.

Structural Organization of Chicken Perlecan—Amino acid sequence alignment demonstrated that human, murine, and chicken perlecan core proteins are highly homologous. A schematic summary of the domain organization of perlecans from human, mouse, chicken, Caenorhabditis elegans, and Drosophila is shown in Fig. 1. The deduced avian protein contains all five domains conserved in the murine molecule (13): domain I (D I), a globular N-terminal domain with putative heparan sulfate (HS) attachment sites; domain II (D II), four copies of LDL receptor ligand-binding (LA) repeats; a central domain III (D III), resembling the short arm of laminin chains with three globules and four cysteine-rich repeats; domain IV (D IV), containing 18 IgG repeats; and domain V (D V), a large globular C-terminal domain resembling part of the G domain of the laminin A chain. Domains II and III are separated by one copy of an Ig repeat-like sequence (IgR1). The domain structure is in accordance with electron microscopy and protein analyses of mammalian perlecans, which indicate that the core protein folds into about 60-80-nm-long linear arrays of globular domains (hence the name perlecan) with extensive secondary structure (23, 24).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1.
Domain structure conservation between perlecans from human, mouse, chicken, C. elegans, and Drosophila. Each gene product (human, huhspg2; murine, mu-hspg2; chicken, gghspg2; D. melanogaster, trol; and C. elegans, unc-52) consists of five consensus domains (D I to D V, top). For simplicity, and because of their considerable size variation and expression of splice variants, they are not drawn to scale, and pairwise alignment scores are only presented where appropriate. The black rectangle represents the signal sequence. In domain I, the presence and number of consensus HS attachment sites, SG(D/E), and of a SEA domain (for details, see "Results"), is indicated by light shading; domain I of the non-vertebrate homologues lack both of these. In domain II, the number of LDL receptor ligand repeats (LA-R) is presented. The dark gray rectangles represent single interspersed Ig repeats; various numbers, as indicated, of such repeats (Ig-R) make up domain IV (the N-CAMs domain). Domain III (laminin A and B short arms) and domain V (globular C terminus of laminin A chain) are also considerably conserved; for further details, refer to "Results." Domains I-III and V, and the single Ig-R between domain II and domain III, were aligned, and the percentage of identical amino acids between hu-hspg2 and mu-hspg2 and mu-hspg2 and gghspg2, respectively, are shown.

 
The N terminus of mature chicken perlecan is presumed to be valine 17, in accordance with the rules of von Heijne (25) and further suggested by alignment with human and murine homologues. The N-terminal portion of domain I (178 amino acids) contains two closely spaced Ser-Gly-Asp/Glu (SGD and SGE) sequences, which are potential attachment sites for HS side chains (26). The C-terminal region of domain I (~90 residues) shows similarities to a conserved sequence called SEA (sperm protein, enterokinase, and agrin) module, which has diverse functions in O-glycosylated proteins (27).

Domain II, characterized by homology to the LDL receptor, is comprised of 199 residues and contains four LA repeats, which are 40-residue-long motifs with clusters of negatively charged residues typically in the sequence Ser-Asp-Glu (SDE), and with 6 cysteines each (28). As in mammalian perlecans, the first LA repeat is separated from the other three by a cysteine-free, proline-rich linker sequence. In chicken perlecan, the linker comprises 38 residues with a proline content of 37%; in human perlecan, it comprises 50 residues (22% proline), and in the murine protein, it comprises 50 residues (26% proline). Shorter linker sequences between two LA repeats are a hallmark property of LA repeat clusters in practically all members of the LR supergene family.

The presence and structure of the single 96-residue Ig-like repeat between domains II and III have been conserved from the chicken to mammals and is also present in the C. elegans homologue (29, 30). The 1168-residue-long domain III contains four subdomains consisting of internal cysteine-rich repeats similar to those typically found in domains III and V of the short arm of the laminin A and B chains (31-38). An Arg-Gly-Asp triplet that confers cell binding (39, 40) found in murine perlecan domain III is absent from the avian protein. Overall, the domain III of the chicken protein is over 60% identical to the primary sequence of murine and human perlecan. Domain IV contains 18 consecutive copies of homologous repeats, which are similar to the Ig repeats in N-CAM (41). Like the single Ig repeat between domain II and III, all of the 18 repeats in domain IV are about 100 residues in length and show all the sequence characteristics ascribed to these motifs (41, 42). With a total of 19 of these repeats, the chicken protein is positioned between murine (total of 15 repeats) and Drosophila (14 repeats total) and human perlecan (22 repeats total) (13, 43). The largest isoform of the C. elegans homologue contains a maximum of 17 Ig repeats (44).

Domain V, termed the laminin A chain-G-like domain (45), resembles the large C-terminal globular G domain of the A chains of laminin (31, 35, 38). The three globular folds and the two EGF-like regions separating them are also highly homologous to the corresponding domains in mammalian perlecans (Fig. 1). In summary, the chicken perlecan protein sequence is a prime example for the conservation of large multidomain proteins from avian species to mammals, likely indicating important function(s), which in fact may be exercised by the individual portions of the large molecule.

Partial Genomic Structure of Chicken Perlecan—We gained insight into the structure of the chicken hspg2 gene from sequence analysis of two clones, f14 and f20. The two clones overlap in an area representing parts of domain I to the beginning of the second cysteine-rich repeat of domain III, span a length of 4831 nucleotides, and cover 15 exons separated by 14 introns (Fig. 2). At the 5'-end, the clones correspond to 5 exons encoding the SEA module at the C terminus of domain I. Following this module, the first and second LA repeats, including the linker motif, are encoded by a single exon, exon 7. The two distal LA repeats of perlecan domain II are each encoded by separate exons, exon 8 and 9 (Fig. 2). In the human LDL receptor gene (46), the structure of the corresponding genomic region is analogous. The Ig repeat following the LA repeats of domain II is encoded by exons 10 and 11 (Fig. 2). The 3'-region defined by the clones specify 5 exons coding for the N-terminal region of domain III. Thus, the comparison of the genomic regions showed a high degree of homology between the human (43) and avian hspg2 genes.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 2.
Comparison of partial genomic structure specifying domains I-III of human and chicken HSPG2. The intron-exon structures of domains I-III (represented by DI-DIII), including the Ig-like repeat 1 (IgR1), of the human and chicken hspg2 genes are compared. The different domains are marked in different colors: domain I (the part representing the SEA domain) is in yellow, domain II (LDL receptor homologous domain) is in red, Ig-like repeat 1 (IgR1) is in orange, and domain III (laminin A short arm domain) is in gray. The numbers inside the colored boxes indicate the exon number, referring to the total number of exons of the entire human hspg2 gene; the numbers above the boxes indicate the number of amino acid residues encoded by the corresponding exon. Note that the intron sizes are not to scale.

 
Expression Pattern of Chicken Perlecan in Various Tissues and at Different Follicular Developmental Stages—Next, we determined the expression of chicken perlecan by Northern blotting (Fig. 3). The vitreous body of the eye, intestine, and testis showed a single ~18-kb message. A hybridization signal of the same size was also observed in mRNA isolated from a pool of follicles with sizes ranging from 3 mm to 1 cm in diameter (Fig. 3, foll.). In skeletal muscle, low levels of transcript were detected, in agreement with previous reports in differentiated myoblasts (47, 48). Inasmuch as the focus of the current study is the development of extracellular matrices in the ovarian follicle, we compared the expression patterns of laminin and agrin with that of perlecan during the life span of a fully differentiated follicle. Fig. 4 shows the results obtained with phase I follicles (termed "white," as they lack yolk), very small (vsw; 1 mm diameter), small (sw; 1-3 mm diameter), or large (lw; 3-5 mm diameter) and early phase II follicles, which contain oocytes that have begun to take up yolk. It is technically difficult to obtain RNA with sufficiently good quality from the yolk-filled larger yellow follicles. However, RNA from the remaining extraoocytic tissue following ovulation (Fig. 4, postovulatory sac (PS)) was isolated 1 (-1d) to 3 days (-3d) after ovulation; thereafter, it is difficult to identify the follicle rudiments. Perlecan expression showed no clear trend during the preovulatory phase, whereas transcript levels of laminin, and particularly of agrin, appeared to decrease with increasing follicle size. In the PS, the levels of all three mRNAs were high for at least 3 days. Based on our previous investigations on follicle development (49, 50), this pattern indicates expression of perlecan by the somatic follicle cells and not by the oocyte. This is furthermore in agreement with the finding of perlecan expression in human ovarian stroma (12). To shed further light on the cellular origin of perlecan in the follicle, we prepared and analyzed mRNA from the theca and granulosa cells of follicles separated by 1 day of growth, from the largest (GC1, Th1) to the smallest follicle from which these cells can be obtained in pure form (GC5, Th5; ~5 days before ovulation) (Fig. 5). Clearly, perlecan is derived exclusively from the theca cells in all follicles. As detailed below, this finding is compatible with the localization of perlecan within the follicle wall.



View larger version (89K):
[in this window]
[in a new window]
  		FIG. 3.
Northern blot analysis of chicken perlecan expression in various tissues. Top panel, total RNA (30 µg of each) from various tissues was separated on a 1% glyoxal-agarose-gel and then transferred to a nylon membrane as described under "Experimental Procedures." F3 follicle (foll.); vitreous body of the eye (eye); intestine (intest.); testis (test.); and skeletal muscle (mus.). The blot was hybridized with the radiolabeled 339-bp fragment corresponding to nucleotides 6872-7210 bp of chicken HSPG2 cDNA, and the autoradiogram was exposed for 36 h. The amounts of RNA loaded were standardized by hybridization with a probe against chicken {beta}-actin (bottom panel). Size markers are indicated on the right.

 



View larger version (63K):
[in this window]
[in a new window]
  		FIG. 4.
Expression analysis of chicken perlecan, chicken laminin, and chicken agrin in ovarian follicles at different developmental stages. Top panels, total RNA (30 µg of each) from very small white follicles (vsw), small white follicles (sw), large white follicles (lw), small yellow follicles (sy), and from postovulatory sac isolated 1 day (PS -1d), 2 days (PS -2d), and 3 days (PS -3d) after ovulation were separated on a 1% glyoxal-agarose-gel and transferred to a nylon membrane as described under "Experimental Procedures." The membrane was hybridized with the radiolabeled 339-bp chicken HSPG2 probe described in the legend for Fig. 3, the chicken agrin probe pBG5 (16), or the 845-bp laminin B1 probe (85). The single transcripts detected had sizes of 18, 10, and 6 kb for perlecan, agrin, and laminin B1, respectively. Bottom panel, the amounts of RNA loaded were standardized by hybridization with a probe against chicken {beta}-actin.

 



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 5.
Expression of chicken perlecan in distinct cell types of follicles at different oocyte growth phases. Top panel, total RNA (30 µg of each) from granulosa cells (GC1-GC5) and theca cells (Th1-Th5) (the numbers indicate the source of cells, i.e. from yellow follicles, F1-F5, in order of decreasing size) was separated on a 1% glyoxal-agarose-gel and transferred to a nylon membrane as described under "Experimental Procedures." The 18-kbp perlecan transcript was detected exclusively in the theca layer cells. Bottom panel, the blot was hybridized with a cDNA probe against chicken {beta}-actin. Size markers are indicated on the right.

 
Expression and Ligand Binding Properties of the Chicken Perlecan LDL Receptor Ligand Binding Repeats—To obtain insights into any hitherto unknown functional properties of perlecan, and based on our interest in proteins containing LDL receptor ligand-binding (LA) repeats, we aimed at expression of correctly folded perlecan domain II. We utilized our recently developed system to express functionally folded clusters of LA repeats, as described for the chicken LDL receptor ligand-binding domain (18). To our knowledge, this domain has not been expressed previously without being linked to neighboring domain(s), nor has it been studied in terms of function (26, 51, 52). We generated a fusion protein between MBP and perlecan LA repeats 1-4 and 2-4 in E. coli. Although the levels of folded LA 1-4 were too low to be useful for further analysis, likely due to the interference by the long proline-rich linker (see nucleotide sequence; the linker region is encoded by a region containing 89% G and C), the fusion protein containing LA 2-4 gave satisfactory levels of correctly folded product (Fig. 6A). We first used the recombinant protein for the production of a highly specific antibody against chicken perlecan. The power of using an MBP·LA cluster fusion protein as antigen has previously been shown in studies on the avian LDL receptor (18). The polyclonal rabbit antibody against chicken perlecan was designated anti-perl2-4 (Fig. 6A). Interestingly, in the C. elegans homologue unc-52, the LDL receptor homology domain consists of only three LA repeats, suggesting that these three repeats can function per se. Indeed, the LA 2-4 protein bound RAP, an established in vitro ligand of all LDL receptor-related proteins (53-55) (Fig. 6B). As shown in Fig. 7, chicken perlecan LA repeats 2-4 also bind plasma VLDL in the fashion typical for bona fide lipoprotein receptors, i.e. with high affinity, dependent on Ca2+ and inhibitable by RAP, and with high affinity as indicated by the attenuated displacement of radiolabeled VLDL by unlabeled autologous competitor. Finally, It was important to show that the perl2-4 domain not only was capable of lipoprotein binding in the solid phase but also in a cellular context. As shown in Fig. 8, receptor-mediated uptake of the apolipoprotein B-containing ligand, LDL, into cells that had been induced to express LDL receptors by sterol depletion was abolished by the addition of the soluble perlecan domain to the incubation medium. Inasmuch as perlecan is found in the extracellular matrix, the inhibition of uptake of the lipoprotein by the cluster of three perlecan LA repeats is compatible with a role of perlecan in lipoprotein retention in the extracellular environment.



View larger version (61K):
[in this window]
[in a new window]
  		FIG. 6.
Analysis of bacterially expressed chicken perlecan ligand-binding domain (perl2-4) and RAP binding function. Perl2-4 was bacterially expressed as a His-tagged fusion protein with maltose-binding protein, purified, and refolded as described under "Experimental Procedures." A, 2 µg of reduced perl2-4 (lanes 1, 3, 5, and 7) and non-reduced perl2-4 (lanes 2, 4, 6, and 8) were stained with Coomassie Blue (lanes 1 and 2) or subjected to immunoblotting with anti-maltose-binding protein (1:1000; lanes 3 and 4), anti-perl2-4 preimmune serum (1: 1000; lanes 5 and 6), or anti-perl2-4 serum (1:1000; lanes 7 and 8). B, inhibition of RAP-Myc binding to perl2-4 with different amounts of GST·RAP. Purified and refolded perl2-4 (2 µg) was subjected to SDS-PAGE under non-reducing conditions followed by transfer to PVDF membrane as described under "Experimental Procedures." PVDF membrane strips were incubated with 10 µg/ml RAP-Myc alone (lane 1) or together with 25 µg/ml (lane 2), 50 µg/ml (lane 3), or 100 µg/ml (lane 4) GST·RAP. Bound RAP-Myc was detected with anti-Myc antibodies, horseradish peroxidase-goat anti-mouse IgG, and ECL. Molecular size markers are indicated.

 



View larger version (57K):
[in this window]
[in a new window]
  		FIG. 7.
Binding of 125I-VLDL to perl2-4, effects of EDTA, GST·RAP, and VLDL. Purified and refolded perl2-4 (2 µg/lane) was subjected to SDS-PAGE under non-reducing conditions followed by transfer to PVDF membrane as described under "Experimental Procedures." The PVDF membrane strips were incubated in buffer containing 1.5 x 106 cpm/ml 125I-VLDL (specific activity, 530 cpm/ng) (shown in C and lanes 1-9). The incubations in lanes 1-3 contained 2 (lane 1), 5 (lane 2), or 10 (lane 3) mM EDTA; in lanes 4-6, 5- (lane 4), 20- (lane 5), or 100-fold (lane 6) excess of GST·RAP; and in lanes 7-9, 2- (lane 7), 5- (lane 8), or 10-fold (lane 9) excess of unlabeled VLDL. Autoradiography was performed by exposure to Kodak X-Omat Blue XB-1 film. Molecular size markers are indicated.

 



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 8.
Perlecan domain II blocks LDL binding to cell surface receptors. 3T3 cells were grown overnight on culture slides in medium supplemented with 10% lipoprotein-deficient serum in the presence of 1 µM of the statin, NK104. The cells were incubated for 1 h at 37 °C in medium plus lipoprotein-deficient serum with 50 µg/ml LDL (A), with 50 µg/ml LDL plus 250 µg/ml perl2-4 (B), or without addition (C). The uptake of LDL was detected with rabbit anti-chicken ApoB antibodies followed by Alexa Fluor 568-labeled anti-rabbit IgG as described under "Experimental Procedures." The nuclei were counterstained with DAPI.

 
Immunohistochemical Localization of Chicken Perlecan and Other Extracellular Matrix Proteins in Testis and Follicles—Next, we analyzed the distribution of extracellular matrix components in rooster testis (Fig. 9). The interstitial space of testes contains a multilayered myofibroblastic complement and relatively few Leydig cells (56). The peritubular space is delineated by a classical basement membrane around the seminiferous tubules. Immunostaining with antibodies against agrin (Fig. 9A), laminin (Fig. 9B), and (mouse) perlecan (Fig. 9C), typical basal basement membrane components, and for collagen I (Fig. 9D), a connective tissue constituent, stained the peritubular structures in more or less continuous patterns, indicating fibrillar or membranous sources which are not entirely congruent. In contrast, the pattern observed with anti-perl2-4 (Fig. 9F) is one of a less organized extracellular matrix material and is not at all like that of the bona fide lamina components agrin and laminin. We further compared perlecan immunostaining to that obtained with different antibodies and at larger magnifications. In Fig. 9G, we used the antibody 3G10 against heparitinase-treated HS side chains (57), and in Fig. 9H, we used our monoclonal antibody 5C9 against chicken perlecan; Fig. 9I is a magnification of the indicated area in panel F. It is obvious that perlecan localization with mAb 5C9 and polyclonal antibody anti-perl2-4 are identical, but very different from the localization of the major antigen(s) of 3G10.



View larger version (152K):
[in this window]
[in a new window]
  		FIG. 9.
Immunofluorescence images of rooster testis showing the distribution of reactivity against agrin (A), laminin B (B), mouse perlecan (C), collagen I (D), perlecan (E, F, H, and I), and heparansulfate stubs (G). Cryostat sections were incubated with anti-agrin (A), mAb 3H11 (B), anti-mouse perlecan (C), mAb COLI (D), anti-perl2-4 preimmune serum (E), anti-perl2-4 (F and I), mAb 3G10 (G), mAb 5C9 (H), and secondary antibodies as described under "Experimental Procedures." Prior to antibody staining, the section in panel G was treated with heparitinase. Panel I shows an enlargement of the area indicated in panel F to facilitate comparison with panel H. Immunoreactive proteins appear in green; the nuclei were counterstained with DAPI.

 
Knowing the staining specificities of the antibodies for the well characterized structures in testis, we analyzed the extracellular matrix in the follicle cell layers (Fig. 10), with a special focus on the membrane between the theca and the granulosa cells. This membrane could be visualized by immunostaining with a rabbit antiserum raised against one of its major components (Fig. 10A).3 The membrane was not stained with anti-agrin (Fig. 10B), anti-laminin (Fig. 10C), anti-fibronectin (Fig. 10D), and the antibody directed to the HS-"stubs", mAb 3G10 (Fig. 10F). The staining with mAb 3G10 is, as expected, very similar to that obtained with mAb 2E9 against syndecan (Fig. 10G), which is one of the main membrane-bound HS proteoglycans. Importantly, although syndecan and the 3G10-antigens are concentrated in the theca interna adjacent to the granulosa cells, perlecan, visualized with anti-perl2-4 (Fig. 10H) and 5C9 (Fig. 10I), is localized almost exclusively in the theca externa, in agreement with the data shown in Fig. 5.



View larger version (88K):
[in this window]
[in a new window]
  		FIG. 10.
Immunofluorescence images of sections of chicken follicle walls showing the distribution of extracellular matrix proteins. Cryostat sections from a 7-mm diameter follicle were stained for ggBM1 (with anti-ggBM1; A), agrin (B), laminin B (mAb 3H11; C), fibronectin (D), heparansulfate stubs (Mab 3G10; E and F), syndecan-1/syndecan-3 (mAb 2E9; G), and perlecan (anti-perl2-4, H; and mAb 5C9, I) as detailed under "Experimental Procedures." Prior to antibody staining, the section shown in panel F, but not in panel E, was treated with heparitinase. Immunoreactive proteins appear in green; the nuclei were counterstained with DAPI.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The molecular characterization of the first perlecan of a non-mammalian vertebrate reveals several novel aspects about the biology of the most common heparan sulfate proteoglycan component of extracellular matrices. As outlined below, these aspects include structure/function relationships, the physiological significance of the specialized extracellular matrix in the avian ovarian follicle, and lipoprotein transport into oocytes. The structural features and their conservation during evolution (Fig. 1) indicate that perlecan function(s) have been maintained, at least from birds to mammals. However, certain differences between chicken perlecan and that of other species are noteworthy. Despite the presence of a single cysteine in domain I, it displays the same degree of identity to murine and human perlecan as the other domains overall. However, the domain I of the avian protein appears to contain only two putative sites for glycan attachment (i.e. closely spaced triplets, SG(E/D)), one less than the murine and human homologues. In perlecans from C. elegans (termed unc-52) and Drosophila melanogaster (gene designation, trol, for terribly reduced optical lobes; Refs. 58 and 59), SEA motifs are absent. Future studies will address whether the consensus serine residues are glycated, and if so, will delineate the nature of the side chains in the avian protein. The features of the remaining domains have been highly conserved in mammalian perlecan molecules, in particular the presence of a single Ig repeat motif (IgR1) between domains II and III.

Perlecan is one of a growing number of proteins containing small clusters of four or fewer LA repeats; LRP5/6 (60, 61) and two proteins in the fertilization envelope and oocytic cortical granules, respectively, of the sea urchin (62, 63) are other examples. Given the possibility that small LA clusters are ligand-binding competent (64, 65), the elucidation of the physiological significance of such "minireceptors" is important. Thus, the domain II of perlecan is of special interest in the context of the involvement of LR gene family members in avian development. This domain contains four closely spaced LA repeats typical for the ligand-binding domains of classical LRs; the N-terminal single repeat is separated from the group of repeats 2-4 by a proline-rich linker, the same arrangement as in mammalian perlecans (13, 66). Interestingly, in the human LDL and VLDL receptor genes, as in the chicken hspg2 gene (Fig. 2), large exons specify contiguous regions consisting of at least two LA repeats plus the linker, whereas other LA repeats are individually encoded by short exons, with introns exactly at the repeat boundaries (46). This conservation may point to a significant requirement for maintenance of the element to preserve important function(s) in birds and mammals. The current findings on the genomic structure (Fig. 2) and on the functional properties of domain II (Figs. 6, 7, 8) strongly suggest that this conserved function is the binding of lipoproteins in the extracellular milieu; this possibility has been previously reported (67). Perlecan might be the product of an ancestral gene that functions in binding lipoproteins or lipidated ligands since LA repeat domains are already present in the perlecans of C. elegans and Drosophila; another such ancestral LR gene may be that for LRP in C. elegans (68). Drosophila perlecan and mammalian megalin, a large LDL receptor gene family member, have been shown to bind hedgehog proteins, cholesterylated signal proteins involved in multiple developmental processes (58, 69). This binding is heparin-insensitive, which indicates that the interaction may be mediated by the domain II of perlecan rather than by the HS chains. Significantly, domain II is present even in the shortest known isoform of unc-52, which lacks domains IV and V altogether (30). Furthermore, domain II of unc-52 consists of only three LA repeats, and again, these are encoded by one long exon specifying two repeats and one short exon, the 5'-part of which encodes the C-terminal third repeat. It is clear from the first detailed functional analysis of this domain that three perlecan LA repeats are entirely sufficient for the binding of apoB-containing lipoproteins such as VLDL (Figs. 7 and 8).

Similar conclusions about the functional requirement for a minimal number of LA repeats have recently been drawn from results of structure/function analysis of the prototype LDL receptor (70). A reliable model at 3.7-Å resolution for the structure of the ligand-binding domain (7 LA repeats) and the EGF precursor domain (consisting of EGF precursor modules A, B, C, and the so-called {beta}-propeller) of the receptor at pH 5.3 has been generated (70). The model predicts that lipoproteins bound at physiological pH to LA repeats are reversibly displaced at the acidic pH in the endosome by the {beta}-propeller subdomain. The LA repeats most critical to this interaction with the {beta}-propeller, and thus likely also for lipoprotein binding, are LA repeats 4 and 5, i.e. those separated by the conserved linker; repeat 5 has been shown by mutational analysis to be particularly critical (71). The presence, already in the avian perlecan gene, of linked LA repeats encoded by a single exon provides evolutionary support for the ligand-displacement model (70). In the future, it will be of interest to examine the significance of the differences both in length and in structure between the linkers in various LRs, in particular in avian perlecan and LDL receptor, which diverge significantly (18). In any case, as outlined below, the localization of perlecan in the follicle wall and its ability to bind VLDL are to be considered in view of further recent reports on the potential role of {beta}-propeller interfaces in protein-protein interaction (72).

In immunological experiments, we demonstrate that the HSPG perlecan is absent from the extracellular matrix separating the thecal cells from the granulosa cell layer. In the absence of molecular details about this membrane, it has been called "follicle basement membrane" or "follicle basal lamina" for over 3 decades (73, 74), implying that it contains obligatory components of basement membranes. However, studies to be reported elsewhere3 have identified the main component of this membranous material as a collagenous acidic proteoglycan that is secreted from the granulosa cells. Indeed, the spatially well defined extracellular material lacks the typical components of basement membranes, including perlecan (Figs. 9 and 10). VLDL particles released from the thecal vasculature would be unable to permeate across a bona fide basal lamina and thus would not become available for receptor-mediated uptake into oocytes. Thus, our studies have resolved this apparent paradox by demonstrating that although perlecan is a major component of follicular extracellular matrix, it is neither part of the purported basement membrane nor synthesized by the adjacent granulosa cells, but by theca externa cells. However, perlecan in the theca externa, in addition to performing its structural function as extracellular matrix protein, might act as a transient holding compartment for VLDL and possibly other serum components destined for yolk uptake. In vivo, the transient ligand binding capacity could undergo changes in a diurnal rhythm, compatible with the well known mode of vitellogenin and VLDL deposition in concentric spherical shells in the oocytic yolk, the number of which is identical to the number of days in which oocytes take up the bulk of yolk (75, 76). VLDL binding, sensitive to Ca2+ levels and pH, could occur to both the LA repeats and the HS chains of perlecan since the latter are also implicated as binding sites for VLDL (77-79). Furthermore, VLDL binding might be mediated or enhanced by changing concentrations of locally secreted lipases (77, 80, 81). Thus, plasma-derived VLDL particles destined for oocyte uptake either may saturate the available binding sites in the extracellular space so as to allow efficient diffusion toward receptors on the oocyte surface or may be bound reversibly, in diurnal rhythm.

In this respect, two recent reports may suggest an alternative mechanism for reversible binding of VLDL to perlecan. These studies at the ultrastructural level show that {beta}-propellers, i.e. YWTD consensus-containing repeat structures present in several proteins including LRs, form interfaces with high affinity for LA repeats of LRs (70) and laminin EGF-like (LE) modules (72). Importantly, perlecan contains both LA and LE domains, and moreover, strongly interacts with nidogen (82-84). In this context, the {beta}-propeller within the globular nidogen domain G3 was shown by Takagi et al. (72) to function as an interface for LEs 3 and 4 of the laminin {gamma}1 subunit. The observed {beta}-propeller interactions are pH-dependent, and at least in the case of the LA repeats, Ca2+-dependent. Thus, although an interaction of the nidogen {beta}-propeller with LA repeats has not been shown, this is a very likely possibility. If such an interaction exists, rhythmic changes in Ca2+ concentrations, pH, or VLDL concentration in the extracellular matrix of the theca externa may induce conformational change(s) in the binding partners, leading to release of VLDL from the LA repeats of perlecan for subsequent uptake into the oocyte.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ584653 [GenBank] .

* This work was supported by Grants FWF; F-606 and F-608 from The Austrian Science Foundation (to W. J. S. and J. N.) and Grant NS33981-02 from the National Institutes of Health (to W. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Institute of Medical Biochemistry, Dept. of Molecular Genetics, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria. Tel.: 43-1-4277-61803; Fax: 43-1-4277-61804; E-mail: wolfgang.schneider{at}univie.ac.at.

1 The abbreviations used are: LR, lipoprotein receptor; LDL, low density lipoprotein; VLDL, very low density lipoprotein; GST, glutathione S-transferase; RAP, receptor-associated protein; PVDF, polyvinylidene difluoride; DAPI, 4',6-diamidino-2-phenylindole; MBP, maltose-binding protein; mAb, monoclonal antibody; HS, heparan sulfate; N-CAM, neural cell adhesion molecule; EGF, epidermal growth factor; TBS, Tris-buffered saline; LE, laminin EGF-like; LA, LDL receptor ligand binding. Back

2 M. Balasubramani, M. E. Bier, S. Hummel, W. J. Schneider, and W. Halfter, submitted. Back

3 S. Hummel, S. Christian, A. Osanger, and W. J. Schneider, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. D. Blaas, Department of Medical Biochemistry, Vienna Biocenter, Austria, Dr. J. Hassell, Shriners Hospital for Children, Tampa, FL, and Dr. G. David, Center of Human Genetics, University of Leuven, Belgium, for gifts of valuable reagents.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bujo, H., Hermann, M., Lindstedt, K. A., Nimpf, J., and Schneider, W. J. (1997) J. Nutr. 127, Suppl. 5, 801S-804S[Medline] [Order article via Infotrieve]
  2. Schneider, W. J., Osanger, A., Waclawek, M., and Nimpf, J. (1998) Biol. Chem. 379, 965-971[Medline] [Order article via Infotrieve]
  3. George, R., Barber, D. L., and Schneider, W. J. (1987) J. Biol. Chem. 262, 16838-16847[Abstract/Free Full Text]
  4. Barber, D. L., Sanders, E. J., Aebersold, R., and Schneider, W. J. (1991) J. Biol. Chem. 266, 18761-18770[Abstract/Free Full Text]
  5. Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175[Medline] [Order article via Infotrieve]
  6. Stifani, S., Barber, D. L., Aebersold, R., Steyrer, E., Shen, X., Nimpf, J., and Schneider, W. J. (1991) J. Biol. Chem. 266, 19079-19087[Abstract/Free Full Text]
  7. Perry, M. M., and Gilbert, A. B. (1979) J. Cell Sci. 39, 257-272[Abstract/Free Full Text]
  8. Nimpf, J., Radosavljevic, M., and Schneider, W. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 906-910[Abstract/Free Full Text]
  9. Perry, M. M., Gilbert, A. B., and Evans, A. J. (1978) J. Anat. 125, 481-497[Medline] [Order article via Infotrieve]
  10. Evans, A. J., Perry, M. M., and Gilbert, A. B. (1979) Biochim. Biophys. Acta 573, 184-195[Medline] [Order article via Infotrieve]
  11. Shen, X., Steyrer, E., Retzek, H., Sanders, E. J., and Schneider, W. J. (1993) Cell Tissue Res. 272, 459-471[CrossRef][Medline] [Order article via Infotrieve]
  12. Murdoch, A. D., Dodge, G. R., Cohen, I., Tuan, R. S., and Iozzo, R. V. (1992) J. Biol. Chem. 267, 8544-8557[Abstract/Free Full Text]
  13. Noonan, D. M., Fulle, A., Valente, P., Cai, S., Horigan, E., Sasaki, M., Yamada, Y., and Hassell, J. R. (1991) J. Biol. Chem. 266, 22939-22947[Abstract/Free Full Text]
  14. Noonan, D. M., Horigan, E. A., Ledbetter, S. R., Vogeli, G., Sasaki, M., Yamada, Y., and Hassell, J. R. (1988) J. Biol. Chem. 263, 16379-16387[Abstract/Free Full Text]
  15. Sambrook, J., Russell, D. W. (2001) in Molecular Cloning: A Laboratory Manual (Argentine, J., ed) 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  16. Tsen, G., Halfter, W., Kroger, S., and Cole, G. J. (1995) J. Biol. Chem. 270, 3392-3399[Abstract/Free Full Text]
  17. Koch, S., Strasser, V., Hauser, C., Fasching, D., Brandes, C., Bajari, T. M., Schneider, W. J., and Nimpf, J. (2002) EMBO J. 21, 5996-6004[CrossRef][Medline] [Order article via Infotrieve]
  18. Hummel, S., Lynn, E. G., Osanger, A., Hirayama, S., Nimpf, J., and Schneider, W. J. (2003) J. Lipid Res. 1, 1
  19. Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139[Abstract/Free Full Text]
  20. Nimpf, J., George, R., and Schneider, W. J. (1988) J. Lipid Res. 29, 657-667[Abstract]
  21. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  22. Halfter, W. (1993) J. Neurosci. 13, 2863-2873[Abstract]
  23. Ledbetter, S. R., Fisher, L. W., and Hassell, J. R. (1987) Biochemistry 26, 988-995[CrossRef][Medline] [Order article via Infotrieve]
  24. Paulsson, M., Yurchenco, P. D., Ruben, G. C., Engel, J., and Timpl, R. (1987) J. Mol. Biol. 197, 297-313[CrossRef][Medline] [Order article via Infotrieve]
  25. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690[Abstract/Free Full Text]
  26. Dolan, M., Horchar, T., Rigatti, B., and Hassell, J. R. (1997) J. Biol. Chem. 272, 4316-4322[Abstract/Free Full Text]
  27. Bork, P., and Patthy, L. (1995) Protein Sci. 4, 1421-1425[Medline] [Order article via Infotrieve]
  28. Herz, J., and Willnow, T. E. (1994) Ann. N. Y. Acad. Sci. 737, 14-19[Medline] [Order article via Infotrieve]
  29. Rogalski, T. M., Williams, B. D., Mullen, G. P., and Moerman, D. G. (1993) Genes Dev. 7, 1471-1484[Abstract/Free Full Text]
  30. Rogalski, T. M., Mullen, G. P., Bush, J. A., Gilchrist, E. J., and Moerman, D. G. (2001) Biochem. Soc. Trans. 29, 171-176[CrossRef][Medline] [Order article via Infotrieve]
  31. Ehrig, K., Leivo, I., Argraves, W. S., Ruoslahti, E., and Engvall, E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3264-3268[Abstract/Free Full Text]
  32. Hunter, D. D., Porter, B. E., Bulock, J. W., Adams, S. P., Merlie, J. P., and Sanes, J. R. (1989) Cell 59, 905-913[CrossRef][Medline] [Order article via Infotrieve]
  33. Montell, D. J., and Goodman, C. S. (1988) Cell 53, 463-473[CrossRef][Medline] [Order article via Infotrieve]
  34. Montell, D. J., and Goodman, C. S. (1989) J. Cell Biol. 109, 2441-2453[Abstract/Free Full Text]
  35. Nissinen, M., Vuolteenaho, R., Boot-Handford, R., Kallunki, P., and Tryggvason, K. (1991) Biochem. J. 276, 369-379[Medline] [Order article via Infotrieve]
  36. Pikkarainen, T., Kallunki, T., and Tryggvason, K. (1988) J. Biol. Chem. 263, 6751-6758[Abstract/Free Full Text]
  37. Sasaki, M., and Yamada, Y. (1987) J. Biol. Chem. 262, 17111-17117[Abstract/Free Full Text]
  38. Sasaki, M., Kleinman, H. K., Huber, H., Deutzmann, R., and Yamada, Y. (1988) J. Biol. Chem. 263, 16536-16544[Abstract/Free Full Text]
  39. Ruoslahti, E., and Pierschbacher, M. D. (1987) Science 238, 491-497[Abstract/Free Full Text]
  40. Chakravarti, S., Horchar, T., Jefferson, B., Laurie, G. W., and Hassell, J. R. (1995) J. Biol. Chem. 270, 404-409[Abstract/Free Full Text]
  41. Cunningham, B. A., Hemperly, J. J., Murray, B. A., Prediger, E. A., Bracken-bury, R., and Edelman, G. M. (1987) Science 236, 799-806[Abstract/Free Full Text]
  42. Williams, A. F., and Barday, A. N. (1988) Annu. Rev. Immunol. 6, 381-405[Medline] [Order article via Infotrieve]
  43. Cohen, I. R., Grassel, S., Murdoch, A. D., and Iozzo, R. V. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10404-10408[Abstract/Free Full Text]
  44. Mullen, G. P., Rogalski, T. M., Bush, J. A., Gorji, P. R., and Moerman, D. G. (1999) Mol. Biol. Cell 10, 3205-3221[Abstract/Free Full Text]
  45. Noonan, D. M., and Hassell, J. R. (1993) Kidney Int. 43, 53-60[Medline] [Order article via Infotrieve]
  46. Sudhof, T. C., Goldstein, J. L., Brown, M. S., and Russell, D. W. (1985) Science 228, 815-822[Abstract/Free Full Text]
  47. Larrain, J., Alvarez, J., Hassell, J. R., and Brandan, E. (1997) Exp. Cell Res. 234, 405-412[CrossRef][Medline] [Order article via Infotrieve]
  48. Villar, M. J., Hassell, J. R., and Brandan, E. (1999) J. Cell. Biochem. 75, 665-674[CrossRef][Medline] [Order article via Infotrieve]
  49. Hirayama, S., Bajari, T. M., Nimpf, J., and Schneider, W. J. (2003) Biol. Reprod. 68, 1850-1860[Abstract/Free Full Text]
  50. Recheis, B., Osanger, A., Haubenwallner, S., Schneider, W. J., and Nimpf, J. (2000) J. Biol. Chem. 275, 35320-35327[Abstract/Free Full Text]
  51. Costell, M., Sasaki, T., Mann, K., Yamada, Y., and Timpl, R. (1996) FEBS Lett. 396, 127-131[CrossRef][Medline] [Order article via Infotrieve]
  52. Groffen, A. J., Buskens, C. A., Tryggvason, K., Veerkamp, J. H., Monnens, L. A., and van den Heuvel, L. P. (1996) Eur. J. Biochem. 241, 827-834[Medline] [Order article via Infotrieve]
  53. Andersen, O. M., Schwarz, F. P., Eisenstein, E., Jacobsen, C., Moestrup, S. K., Etzerodt, M., and Thogersen, H. C. (2001) Biochemistry 40, 15408-15417[CrossRef][Medline] [Order article via Infotrieve]
  54. Bu, G., Geuze, H. J., Strous, G. J., and Schwartz, A. L. (1995) EMBO J. 14, 2269-2280[Medline] [Order article via Infotrieve]
  55. Bu, G. (2001) Int. Rev. Cytol. 209, 79-116[Medline] [Order article via Infotrieve]
  56. Aire, T. A. (1997) Onderstepoort. J. Vet. Res. 64, 291-299[Medline] [Order article via Infotrieve]
  57. David, G., Lories, V., Decock, B., Marynen, P., Cassiman, J. J., and Van den Berghe, H. (1990) J. Cell Biol. 111, 3165-3176[Abstract/Free Full Text]
  58. Park, Y., Rangel, C., Reynolds, M. M., Caldwell, M. C., Johns, M., Nayak, M., Welsh, C. J., McDermott, S., and Datta, S. (2003) Dev. Biol. 253, 247-257[CrossRef][Medline] [Order article via Infotrieve]
  59. Voigt, A., Pflanz, R., Schafer, U., and Jackle, H. (2002) Dev. Dyn. 224, 403-412[CrossRef][Medline] [Order article via Infotrieve]
  60. Brown, S. D., Twells, R. C., Hey, P. J., Cox, R. D., Levy, E. R., Soderman, A. R., Metzker, M. L., Caskey, C. T., Todd, J. A., and Hess, J. F. (1998) Biochem. Biophys. Res. Commun. 248, 879-888[CrossRef][Medline] [Order article via Infotrieve]
  61. Hey, P. J., Twells, R. C., Phillips, M. S., Yusuke, N., Brown, S. D., Kawaguchi, Y., Cox, R., Guochun, X., Dugan, V., Hammond, H., Metzker, M. L., Todd, J. A., and Hess, J. F. (1998) Gene (Amst.) 216, 103-111[CrossRef][Medline] [Order article via Infotrieve]
  62. Wessel, G. M. (1995) Dev. Biol. 167, 388-397[CrossRef][Medline] [Order article via Infotrieve]
  63. Wessel, G. M., Conner, S., Laidlaw, M., Harrison, J., and LaFleur, G. J., Jr. (2000) Biol. Reprod. 63, 1706-1712[Abstract/Free Full Text]
  64. Bajari, T. M., Lindstedt, K. A., Riepl, M., Mirsky, V. M., Nimpf, J., Wolfbeis, O. S., Dresel, H. A., Bautz, E. K., and Schneider, W. J. (1998) Biol. Chem. 379, 1053-1062[Medline] [Order article via Infotrieve]
  65. Obermoeller-McCormick, L. M., Li, Y., Osaka, H., FitzGerald, D. J., Schwartz, A. L., and Bu, G. (2001) J. Cell Sci. 114, 899-908[Abstract]
  66. Kallunki, P., and Tryggvason, K. (1992) J. Cell Biol. 116, 559-571[Abstract/Free Full Text]
  67. Fuki, I. V., Iozzo, R. V., and Williams, K. J. (2000) J. Biol. Chem. 275, 25742-25750[Abstract/Free Full Text]
  68. Yochem, J., and Greenwald, I. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4572-4576[Abstract/Free Full Text]
  69. McCarthy, R. A., Barth, J. L., Chintalapudi, M. R., Knaak, C., and Argraves, W. S. (2002) J. Biol. Chem. 277, 25660-25667[Abstract/Free Full Text]
  70. Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., and Deisenhofer, J. (2002) Science 298, 2353-2358[Abstract/Free Full Text]
  71. Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688[Abstract/Free Full Text]
  72. Takagi, J., Yang, Y., Liu, J. H., Wang, J. H., and Springer, T. A. (2003) Nature 424, 969-974[CrossRef][Medline] [Order article via Infotrieve]
  73. Evans, A. J., and Burley, R. W. (1987) J. Biol. Chem. 262, 501-504[Abstract/Free Full Text]
  74. Gilbert, A. B., Evans, A. J., Perry, M. M., and Davidson, M. H. (1977) J. Reprod. Fertil. 50, 179-181[Abstract/Free Full Text]
  75. Ito, Y., Kihara, M., Nakamura, E., Yonezawa, S., and Yoshizaki, N. (2003) Zoolog. Sci. (Tokyo) 20, 717-726
  76. Romanoff, A. L., Romanoff, A. J. (1949) The Avian Egg, John Wiley and Sons Inc., New York
  77. Mulder, M., Lombardi, P., Jansen, H., van Berkel, T. J., Frants, R. R., and Havekes, L. M. (1993) J. Biol. Chem. 268, 9369-9375[Abstract/Free Full Text]
  78. Ji, Z. S., Brecht, W. J., Miranda, R. D., Hussain, M. M., Innerarity, T. L., and Mahley, R. W. (1993) J. Biol. Chem. 268, 10160-10167[Abstract/Free Full Text]
  79. Wilsie, L. C., and Orlando, R. A. (2003) J. Biol. Chem. 278, 15758-15764[Abstract/Free Full Text]
  80. Fuki, I. V., Blanchard, N., Jin, W., Marchadier, D. H., Millar, J. S., Glick, J. M., and Rader, D. J. (2003) J. Biol. Chem. 278, 34331-34338[Abstract/Free Full Text]
  81. Schneider, W. J., Carroll, R., Severson, D. L., and Nimpf, J. (1990) J. Lipid Res. 31, 507-513[Abstract]
  82. Hopf, M., Gohring, W., Kohfeldt, E., Yamada, Y., and Timpl, R. (1999) Eur. J. Biochem. 259, 917-925[Medline] [Order article via Infotrieve]
  83. Kvansakul, M., Hopf, M., Ries, A., Timpl, R., and Hohenester, E. (2001) EMBO J. 20, 5342-5346[CrossRef][Medline] [Order article via Infotrieve]
  84. Hopf, M., Gohring, W., Mann, K., and Timpl, R. (2001) J. Mol. Biol. 311, 529-541[CrossRef][Medline] [Order article via Infotrieve]
  85. O'Rear, J. J. (1992) J. Biol. Chem. 267, 20555-20557[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
A. Dichlberger, L. A. Cogburn, J. Nimpf, and W. J. Schneider
Avian apolipoprotein A-V binds to LDL receptor gene family members
J. Lipid Res., July 1, 2007; 48(7): 1451 - 1456.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Hummel, S. Christian, A. Osanger, H. Heid, J. Nimpf, and W. J. Schneider
Identification of a Novel Chondroitin-sulfated Collagen in the Membrane Separating Theca and Granulosa Cells in Chicken Ovarian Follicles: THE GRANULOSA-THECA CELL INTERFACE IS NOT A BONA FIDE BASEMENT MEMBRANE
J. Biol. Chem., March 16, 2007; 282(11): 8011 - 8018.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
W. Halfter, U. Winzen, P. N. Bishop, and A. Eller
Regulation of eye size by the retinal basement membrane and vitreous body.
Invest. Ophthalmol. Vis. Sci., August 1, 2006; 47(8): 3586 - 3594.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/22/23486    most recent
M312694200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hummel, S.
Right arrow Articles by Schneider, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hummel, S.
Right arrow Articles by Schneider, W. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement