ARTICLE |
Correspondence to: Mark A. Gibson, Dept. of Pathology, University of Adelaide, South Australia 5005, Australia.
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Summary |
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MP78/70 is a matrix protein, with 78-kD and 70-kD isoforms, which was initially identified in bovine tissue extracts designed to solubilize elastin-associated microfibrils. Peptide analysis has shown that MP78/70 is closely related to the human protein, ßig-h3. In the present study an antibody raised to a synthetic ßig-h3 peptide was shown specifically to identify MP78/70 in purified form and in bovine tissue extracts. This is consistent with MP78/70 and ßig-h3 being the bovine and human forms, respectively, of the same protein. The antibody was further affinity-purified on MP78/70 bound to Sepharose and used to localize the protein in a range of bovine tissues. Immunofluorescence showed that MP78/70 was localized to collagen fibers in tissues such as developing nuchal ligament, aorta and lung, and mature cornea; to reticular fibers in fetal spleen; and to capsule and tubule basement membranes in developing kidney. No general localization to elastic fibers was observed. The staining pattern in most tissues more closely resembled that of Type VI collagen, which occurs as collagen fiber-associated microfibrils, than that of fibrillin-1, a component of elastin-associated microfibrils. However, MP78/70 appeared to be less widely distributed than Type VI collagen. Immunoelectron microscopy showed that MP78/70 was predominantly found in loose association with collagen fibers in most tissues examined and was also located on the surface of the capsule basement membrane in developing kidney. Double labeling experiments indicated that MP78/70 is co-distributed with Type VI collagen microfibrils located in these regions. In some elastic tissues significant immunolabel was detected in regions of interface between collagen fibers and fibrillin-containing microfibrils of adjacent elastic fibers, and at the outer margins of the latter structures. Overall, the evidence points to MP78/70 having a bridging function, perhaps in association with Type VI collagen microfibrils, linking or stabilizing the interaction between interstitial collagen fibrils and other matrix structures, including some basement membranes and elastin-associated microfibrils. (J Histochem Cytochem 45:1683-1696, 1997)
Key Words: MP78/70, ßig-h3, fibrillin-1, Type VI collagen, immunoelectron microscopy, immunofluorescence, bovine tissues
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Introduction |
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We have previously described the isolation and partial characterization of MP78/70, a matrix protein occurring as 78-kD and 70-kD isoforms, from a developing elastic tissue, the nuchal ligament of fetal calves (
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Materials and Methods |
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Antibody Production
A synthetic peptide TQLYTDRTEKLRPEMEG(C) was commercially prepared (Chiron Mimotopes; Melbourne, Australia) that corresponds to residues 118-134 of the proteins encoded by mouse and human ßig-H3 cDNAs. This region of the molecule lies between the cysteine-rich N-terminal domain and the four regions of internal homology that have similarity to the insect adhesion molecules, the fasciclins (
Assessment of Antibody Specificity
The affinity-purified anti-MP78/70 antibodies were assessed by ELISA and immunoblotting for crossreactivity with other matrix proteins (
Immunofluorescence Microscopy
Tissue blocks were taken from adult cattle or 210-day-old fetal calves within 1 hr of maternal death and snap-frozen in OCT compound. Sections (4 µm thick) were cut using a cryostat and incubated with primary antibody, followed by an appropriate secondary antibody coupled to fluorescein as described previously (
Antibodies to Type VI collagen and tropoelastin were applied to unfixed tissues. Affinity-purified rabbit antibodies were applied at a concentration of 20 µg/ml and the monoclonal antibodies used were ascites fluid diluted 1:100. Control sections were incubated with IgG (20 µg/ml) purified from preimmune rabbit serum and diluted ascites containing a monoclonal antibody to the parasite Giardia lamblia (
Immunoelectron Microscopy
For single immunolabeling experiments, blocks approximately 1 mm3 were cut from a range of tissues of a 230-day-old fetal calf and an adult cow within 30 min of death. The tissue blocks were fixed and embedded in either LR White or Lowicryl resin as described previously (
For double labeling experiments, the method was again based on that described by
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Results |
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Antibody Specificity
The affinity-purified anti-[ßig-h3 peptide] antibody reacted strongly with MP78/70 by ELISA, with a titer of 1:320. No crossreactivity of the antibody was detected with elastic fiber-associated proteins, fibrillin-1, MAGPs 1 and 2, and tropoelastin, or with collagen Types I, III, V and VI (not shown). Immunoblotting showed that the antibody reacted with both the 78-kD and the 70-kD isoforms of MP78/70 (Figure 1B, Lane 2). This indicated that a sequence similar to that of the peptide was present in MP78/70. Again, no crossreaction was detected with fibrillin-1, MAGP-1, MAGP-2, or tropoelastin (Figure 1A and Figure 1B). Purified decorin, biglycan, and collagen Types I, III, V, and VI were also shown to be unreactive with the antibody using the immunoblotting technique (data not shown). The anti-[ßig-h3 peptide] antibody was also tested for crossreactivity with other tissue antigens by immunoblotting against a series of sequential extracts from fetal nuchal ligament. The antibody detected only MP78/70 in each of the extracts (Figure 1C). A major portion of MP78/70 was extracted from the tissue by treatment with 6 M GuHCl in the absence of a reducing agent (Figure 1C, Lane 8). However, significant amounts of MP78/70 were extracted only after treatment of the homogenate with a reducing agent, indicating that a proportion of the MP78/70 was disulfide-bonded to an insoluble component of the tissue, consistent with previous observations (
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Immunofluorescence
The distribution of MP78/70 was determined in a range of elastic and nonelastic tissues from a 210-day-old fetal calf and in some adult bovine tissues. In each tissue the MP78/70 distribution was compared to those of Type VI collagen and fibrillin-1. Unless otherwise stated, photographs of control sections incubated with preimmune rabbit IgG or mouse ascites fluid showed no fluorescence signal when the appropriate exposure times were used (not shown).
Elastic Tissues. In developing nuchal ligament, MP78/70 was localized extensively throughout the extracellular matrix (Figure 2A). The staining pattern had extensive overlap with that of Type VI collagen (Figure 2B), which is associated with collagen fibers between cells and elastic fibers. However, some MP78/70 also appeared to be co-localized with fibrillin-1, which is located only on elastic fibers (Figure 2C). In aorta, MP78/70 was localized most strongly in the inner media to the matrix between elastic fibers (Figure 2D). In the outer media, immunostaining was also evident around the surface of smooth muscle cells in the vicinity of basement membranes. The adventitia showed weak staining that appeared to co-distribute with collagen fibers (not shown). A similar distribution was found for Type VI collagen (Figure 2E), which contrasted with the elastic fiber staining of the anti-fibrillin 1 antibody (Figure 2F).
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In lung, MP78/70 was localized as a continuous network throughout the parenchymal tissue. The protein was also identified surrounding bronchioles and in the walls of blood vessels (Figure 3A). This distribution was similar to that of Type VI collagen (Figure 3B). In contrast, anti-fibrillin-1 monoclonal antibody A5 localized in the parenchyma only to elastic fibers, which have a fragmented appearance. Fibrillin-1 was also identified around bronchioles and in the elastic fibers of blood vessel walls (Figure 3C).
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In skin, MP78/70 exhibited a diffuse distribution in the matrix between the elastic fibers throughout the tissue and was particularly prominent in the upper papillary dermis (Figure 3D). Staining of the epidermis and around hair follicles was also evident but this appears to be nonspecific because these structures were also stained in control sections treated with preimmune IgG and photographed with the same exposure time (Figure 3G). The anti-Type VI collagen antibodies gave a strong uniform but diffuse staining pattern throughout the dermal matrix (Figure 3E), whereas anti-fibrillin-1 antibodies localized only to discrete elastic fibers and fine fibrils (Figure 3F). In elastic ear cartilage, MP78/70 was strongly immunolocalized to fibers, presumably collagenous, within the perichondrium and to the outer margins of the cartilage. No staining of the elastic fibers in the cartilage matrix was detected. Under the same experimental conditions these structures stained very strongly with anti-tropoelastin antibodies (results not shown).
Other Tissues. In spleen, MP78/70, Type VI collagen, and fibrillin-1 were all strongly localized in a reticular network that extended throughout the red and white pulp (Figure 4A-C). However, in the fibroelastic septa, MP78/70 and Type VI collagen were distributed in the matrix around the elastic fibers, whereas fibrillin-1 was located only on these structures. MP78/70 was particularly prominent at the interface between the septa and the surrounding pulp (Figure 4A). The most marked difference between the localization of MP78/70 and Type VI collagen occurred in developing skeletal muscle. The anti-MP78/70 antibody gave only weak perimysial staining (Figure 4D) in contrast to antibodies to Type VI collagen and fibrillin-1, both of which gave strong staining of the perimysium and endomysium (Figure 4E and Figure 4F).
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In developing kidney, MP78/70 was identified predominantly in close association with the basement membranes of Bowman's capsule and the proximal tubules. Weak immunostaining of the glomerular mesangium was also evident (Figure 5A). In contrast, Type VI collagen had strong localization throughout the peritubular matrix as well as in the mesangium (Figure 5B). Fibrillin-1 was identified mainly in the mesangium, although some intermittent staining around tubules was evident (Figure 5C). In adult kidney, all three proteins had extensive distributions in the peritubular matrix (Figure 5D-F). The staining intensity of the mesangium relative to the peritubule region varied between the three antibodies. Anti-MP78/70 antibody stained the mesangium weakly (Figure 5D), anti-Type VI collagen antibody stained both regions with comparable intensity (Figure 5E), and anti-fibrillin-1 antibody gave a much stronger signal in the mesangium (Figure 5F).
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In the adult eye, the suspensory ligament of the lens did not stain with anti-MP78/70 or anti-Type VI collagen antibodies (Figure 6A and Figure 6B), in contrast to anti-fibrillin antibodies, which stained this structure intensely (Figure 6C). All three antibodies showed strong staining of the matrix of the ciliary body to which the zonule is attached. In cornea, the anti-MP78/70 and anti-Type VI collagen antibodies both gave intense, diffuse staining throughout the matrix (Figure 6D and Figure 6E), whereas anti-fibrillin-1 antibody localized to discrete fibrils (Figure 6F). No staining of the epithelium was detected with any of the antibodies. Overall, MP78/70 had a distribution that was similar but not identical to that of Type VI collagen in most of the tissues examined, and which was distinct from that of fibrillin-1.
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Immunoelectron Microscopy
The ultrastructural location of MP78/70 was determined in several fetal tissues and adult cornea using the immunogold labeling technique (Figure 7 and Figure 8). In developing nuchal ligament, MP78/70 was predominantly localized in loose association with collagen fibrils (Figure 7A). The localization appeared to be to fine filaments surrounding the collagen fibrils rather than to the fibrils themselves. Significant immunogold labeling was also found in regions of interface between collagen fibers and fibrillin-containing microfibrils of adjacent elastic fibers (Figure 7B). Occasionally immunolabel was detected on fibrillin-containing microfibrils at the outer margins of developing elastic fibers, with no collagen fibrils in close proximity (Figure 7C), suggesting that some MP78/70 was bound to the microfibrils. In control sections incubated with IgG from preimmune rabbit serum as primary antibody, very few immunogold particles were detected and these appeared to be randomly scattered throughout the tissue (Figure 7D). Anti-MP78/70 antibody also localized predominantly around collagen fibers in developing aorta and skin and in mature cornea (Figure 8). Figure 8A is a section of inner aortic media showing extensive labeling around a collagen fiber. Again, no general staining of elastic fibers is evident, although a significant number of immunogold particles are located at the interface with an adjacent elastic fiber. Figure 8B shows a section of upper papillary dermis, the region of skin that was stained most significantly by immunofluorescence. Here, too, MP78/70 is located around bundles of collagen fibrils. Similarly, in adult cornea MP78/70 is immunolocalized in widespread association with the dense collagen fibers that make up the bulk of the tissue (Figure 8C). The Bowman's capsule of developing kidney is shown in Figure 8D. In this region, MP78/70 is localized to fine filaments attached to the outer surface of the capsule basement membrane as well as filaments that surround collagen fibers in the pericapsular connective tissue. No staining of the basement membrane itself is evident. Control sections from the above tissues that had been treated with IgG from preimmune rabbit serum in place of primary antibody showed no significant immunogold labeling (not shown).
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Double labeling experiments were conducted on sections of developing nuchal ligament tissue using pre-embedding labeling with anti-Type VI collagen antibodies followed by postembedding labeling with anti-MP78/70 antibodies. Extensive co-localization of Type VI collagen, identified by protein A-20-nm gold particles, and MP78/70, identified by protein A-10-nm gold particles, was observed to fine microfibrils, characteristic of Type VI collagen, surrounding bundles of collagen fibrils (Figure 9A). Controls sections incubated with IgG from preimmune serum instead of the two antibodies showed no localization of gold particles to these structures (not shown). An additional control was conducted in which the blocks were pre-embedding-labeled for Type VI collagen but the anti-MP78/70 antibodies were omitted from the postembedding treatment (Figure 9B). In this control, Type VI collagen microfibrils were identified by 20-nm gold particles but no labeling of these structures with 10-nm gold was evident. This finding indicates that the protein A-10-nm gold, applied after embedding, is not binding to the anti-Type VI collagen antibodies embedded in the tissue. Overall, the results confirmed that MP78/70 is closely associated with Type VI collagen microfibrils in nuchal ligament tissue.
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Discussion |
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Previous studies have shown that MP78/70 is a significant component of and is strongly bound to the extracellular matrix in developing elastin-rich tissues (
In the present study, antibodies prepared to a synthetic ßig-h3 peptide were used to confirm that MP78/70 is the bovine form of the same protein and to determine its tissue distribution and ultrastructural location within the matrix. MP78/70 was the only protein from a range of purified bovine matrix macromolecules that reacted to the peptide antibody on ELISA and immunoblotting. Moreover, MP78/70 was also the only protein identified by the antibody in extracts of nuchal ligament tissue (Figure 1). Immunofluorescence localization studies showed that MP78/70 was extensively present in the extracellular matrix of a wide range of developing and mature tissues (Figure 2 Figure 3 Figure 4 Figure 5 Figure 6). The protein was identified predominantly in the region of interstitial collagen fibers and in association with some basement membranes, including those around smooth muscle cells in developing aorta and those of Bowman's capsule and tubules in fetal kidney. No general co-localization with elastic fibers was observed. MP78/70 had extensive co-distribution with Type VI collagen in tissues such as developing nuchal ligament, aorta and lung, and mature cornea. However, there were differences in distribution in some tissues, such as developing kidney, skin, and muscle. The most marked difference was observed in fetal skeletal muscle, in which little MP78/70 was detected, in contrast to Type VI collagen, which was found throughout the perimysium and endomysium. Type VI collagen has previously been reported to be a constituent of perimysium and endomysium in skeletal muscle from adult chickens (
Immunoelectron microscopy revealed that MP78/70 was predominantly localized in loose association with collagen fibril bundles in a number of developing tissues and in mature cornea (Figure 7 and Figure 8). In addition, localization of MP78/70 was confirmed at the outer margin of the basement membrane of Bowman's capsule in developing kidney. In fetal nuchal ligament and aorta, the protein was also detected at regions of interface between collagen fibers and the microfibrillar component of developing elastic fibers. In nuchal ligament, some immunostaining for MP78/70 was present at the outer margins of the elastin-associated microfibrils, often in regions in which collagen fibrils were not evident. This indicates that some MP78/70 may be attached to these fibrillin-containing microfibrils.
Double labeling experiments (Figure 9) confirmed that the MP78/70 localization around collagen fibers corresponds to the region containing the fine (5-nm diameter) Type VI collagen microfibrils that surround interstitial collagen fibrils in the extracellular matrix of most tissues (
Overall, the evidence suggests that MP78/70 (ßig-h3) functions as a bridging molecule, possibly in association with Type VI collagen microfibrils, linking interstitial collagen fibers with other structural elements of the extracellular matrix, including certain basement membranes and elastin-associated microfibrils. Such a role may be important for anchoring elastin-associated microfibrils to collagen fibers during stretching of elastic fibers.
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Acknowledgments |
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Supported by the National Health and Medical Research Council of Australia.
We are indebted to Denise Yeats and Betty Reinboth for skilled technical assistance.
Received for publication November 27, 1996; accepted June 9, 1997.
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