Mark A. Gibson

Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils.

Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of the guanidine-solubilized LTBP-2 appeared to be monomeric, indicating that it was not involved in disulfide-bonded aggregation either with itself or with latent TGF-beta. Additional LTBP-2 was resistant to solubilization with 6 M guanidine but was readily extracted with a reductive saline solution. This treatment is relatively specific for solubilization of microfibrillar constituents including fibrillin 1 and microfibril-associated glycoprotein. Therefore, it can be inferred that some LTBP-2 is bound covalently to the microfibrils by reducible disulfide linkages. The evidence suggests that LTBP-2 has a direct role in elastic fiber structure and assembly which may be independent of its growth factor-binding properties. Thus, LTBP-2 appears to share functional characteristics with both LTBP-1 and fibrillins.

Further Characterization of Proteins Associated with Elastic Fiber Microfibrils Including the Molecular Cloning of MAGP-2 (MP25)

Together with the 31-kDa microfibril-associated glycoprotein (MAGP), four polypeptides designated MP340 (340 kDa), MP78 (78 kDa), MP70 (70 kDa), and MP25 (25 kDa) have previously been identified in tissue extracts designed specifically to solubilize the microfibrillar component of elastic fibers. In the present study, both MP78 and MP70 were shown to be forms of a protein which is closely related to the human protein βig-h3, and MP340 was confirmed to be the bovine form of fibrillin-1. Peptide sequences from MP25 proved to be unique, and affinity-purified anti-MP25 antibodies were shown, by immunofluorescence and immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. This confirmed that MP25 was a distinct component of these structures. Expression screening of nuchal ligament cDNA libraries yielded a cDNA, cM10A (770 base pairs) which encodes amino acid sequences matching those of the MP25 peptides. Further library screening with cM10A identified cDNAs which encode the complete primary structures of bovine and human MP25. Bovine and human MP25 were found to be around 80% homologous and contain 170 and 173 amino acids, respectively. Data base searches revealed that MP25 had significant similarity of structure only with MAGP, indicating that the two proteins form a new family of microfibrillar proteins. In acknowledgment, MP25 has been formally renamed MAGP-2, and MAGP is referred to as MAGP-1. The close similarity between the two proteins (57%) is confined to a central region of 60 amino acids where there is precise alignment of 7 cysteine residues. Elsewhere the MAGP-2 molecule is rich in serine and threonine residues and contains an RGD motif. MAGP-2 lacks the proline-, glutamine-, and tyrosine-rich sequences and a hydrophobic carboxyl terminus, characteristic of MAGP-1. These structural differences suggest that MAGP-2 has some functions which are distinct from those of MAGP-1. The locus of the human MAGP-2 gene was identified on chromosome 12 in the region of 12p12.3-12p13.1.

Microfibril-associated glycoprotein-1 (MAGP-1) is specifically located on the beads of the beaded-filament structure for fibrillin-containing microfibrils as visualized by the rotary shadowing technique.

This study used immunoelectron microscopic techniques to define the ultrastructural location of MAGP-1 on the fibrillin-containing microfibrils of the ocular zonule. A specific anti-MAGP-1 monoclonal antibody (MAb), 11B, was produced that did not crossreact with fibrillin-1 or other microfibrillar proteins. MAb 11B was shown by immunofluorescence to localize intensely to zonular tissue. Postembedding immunoelectron microscopy showed that MAGP-1 was associated with microfibrils throughout the zonule, with the exception of a narrow band of microfibrils at the junction with the lens capsule. With preembedding labeling, the anti-MAGP-1 MAb was found to localize in a crossbanding pattern, at intervals of about 50 nm, to microfibrils throughout the zonule and along bundles of microfibrils in surrounding vitreous tissue. Rotary shadowing of isolated microfibrils showed a beads on a string morphology with a periodicity of about 50 nm. With immunogold labeling, the anti-MAGP-1 antibody specifically localized on the beads in a symmetrical manner. Occasionally two gold partides were attached to the same bead, suggesting that multiple MAGP-1 molecules were present in the structure. The results indicate that MAGP-1 is intimately and regularly associated with the bead regions of fibrillin-containing microfibrils. The findings are consistent with a major structural role for MAGP-1 in microfibril biology.

Microfibril-associated Glycoprotein-1 (MAGP-1) Binds to the Pepsin-resistant Domain of the α3(VI) Chain of Type VI Collagen

The interactions of type VI collagen have been investigated, using solid phase binding assays, with two components of the fibrillin-containing microfibrils, the elastin-binding protein, MAGP-1 and its structural relative MAGP-2. Both native and pepsin-treated forms of type VI collagen specifically bound to MAGP-1 but not to MAGP-2. Pepsin type VI collagen was shown to block the binding of MAGP-1 to native type VI collagen indicating that the major MAGP-1-binding site was in the triple-helical region of the molecule. MAGP-1 was found not to bind to collagens I, III, and V. Affinity blotting of pepsin-treated type VI collagen showed that MAGP-1 binding was specific for the collagenous domain of the α3(VI) chain. Decorin and biglycan were found not to inhibit the interaction of pepsin-treated type VI collagen with MAGP-1, indicating that its binding site on the collagen is not close to that for the proteoglycans. Reduction and alkylation of disulfide bonds in MAGP-1 did not destroy its type VI collagen-binding properties, indicating that the binding site was likely to be in the cysteine-free, N-terminal domain of MAGP-1. Interestingly, the interaction of MAGP-1 with type VI collagen was inhibited by tropoelastin, suggesting that the binding sites for tropoelastin and type VI collagen may be in the same domain of MAGP-1. A peptide, corresponding to amino acids 29–38 of MAGP-1, was found to inhibit the interactions of MAGP-1 with type VI collagen and tropoelastin. The results suggest that the peptide may contain the binding sequences for both type VI collagen and tropoelastin, and thus that these two proteins may share the same binding site on MAGP-1. The interactions of MAGP-1 with type VI collagen and tropoelastin were both determined to be of moderately high affinity, withK d values of 5.6 × 10−7 m and 2.6 × 10−7 m, respectively. The findings indicate that MAGP-1 may mediate a molecular interaction between type VI collagen microfibrils and fibrillin-containing microfibrils, structures which are often found in close proximity to each other in a wide range of extracellular matrices.

Microfibril-associated Glycoprotein-2 (MAGP-2) Is Specifically Associated with Fibrillin-containing Microfibrils but Exhibits More Restricted Patterns of Tissue Localization and Developmental Expression Than Its Structural Relative MAGP-1

SUMMARY We developed an affinity-purified anti-MAGP-2 peptide antibody that specifically identified MAGP-2 on Western blots of purified matrix proteins and extracts of nuchal ligament. Immunolocalization studies on tissues from a 210-day-old fetus and a mature bovine showed that MAGP-2 was located in similar regions to MAGP-1 and fibrillin-1 but that the distribution of MAGP-2 was more restricted. In fetal nuchal ligament, skeletal muscle, and spleen the distribution of MAGP-2 was indistinguishable from that of MAGP-1. In contrast to MAGP-1, MAGP-2 was not detected in the medial layer of fetal thoracic aorta and in much of the peritubular matrix of fetal and mature kidney and in the mature ocular zonule. Some differences in the immunolocalization patterns were also evident in fetal lung, cartilage, skin, and heart. Immunoelectron microscopy confirmed that MAGP-2 was specifically associated with fibrillin-containing microfibrils in nuchal ligament, dermis, adventitia of aorta, glomerular mesangium and perimysium. Northern blotting of RNA from tissues of a 210-day-old fetus indicated that steady-state MAGP-2 mRNA levels were highest in nuchal ligament. Significant expression was also detected in lung, heart, skeletal muscle, skin, and Achilles tendon. The tissue pattern of MAGP-2 expression differed significantly from that of MAGP-1. MAGP-2 expression appeared to be higher in nuchal ligament, heart, and skeletal muscle and lower in aorta and kidney. In nuchal ligament, MAGP-2 mRNA expression appeared to peak around 180 days of fetal development, which correlates with the period of onset of elastinogenesis in this tissue. Overall, the immunolocalization and expression patterns of MAGP-2 appeared to be distinct from those of other microfibrillar components. This is consistent with the view that MAGP-2 plays a unique role in the biology of the microfibrils, perhaps by mediating their interaction with cell surfaces at specific stages of development and differentiation.

Fibrillin-1 and fibrillin-2 show temporal and tissue-specific regulation of expression in developing elastic tissues.

The recent characterization of multiple fibrillin genes raises the question of whether each of the fibrillin proteins is a component of elastic fiber microfibrils and whether their expression during development of elastic tissues is consistent with a function associated with elastogenesis. To address these possibilities, the expression of fibrillin-1 and fibrillin-2 was compared with expression of MAGP and tropoelastin in two elastogenic tissues that undergo different developmental programs. For both fibrillins, the greatest increase in expression occurred during the last half of fetal development when elastin production is highest. In fetal bovine nuchal ligament, mRNA levels for fibrillin-1 and fibrillin-2 increased approximately threefold during this period, whereas tropoelastin increased 20-fold. Although the relative increase in expression of both fibrillins was equivalent, the basal level of fibrillin-1 expression was greater than fibrillin-2. In developing bovine aorta, fibrillin mRNA levels again paralleled tropoelastin expression although, compared to ligament, elastin synthesis began at an earlier fetal age in this tissue. Furthermore, the relative increase in aortic fibrillin-2 expression was greater than that for fibrillin-1 and the ratio of fibrillin-2 to fibrillin-1 was higher than in the ligament. In contrast to the fibrillins, MAGP expression in nuchal ligament and aorta remained at a constant high level throughout the fetal period. Indirect immunofluorescent staining and immunoelectron microscopy localized both fibrillins as well as MAGP to elastic fiber microfibrils in these developing tissues. The coordinate upregulation of fibrillin-1 and fibrillin-2 expression with the onset of tropoelastin production is consistent with a role in elastic fiber assembly. Our findings also suggest temporal and tissue-specific regulation for the fibrillins during development.

Microfibril-associated glycoprotein-2 specifically interacts with a range of bovine and human cell types via αVβ3 integrin

Microfibril-associated glycoprotein (MAGP)-1 and MAGP-2 are small structurally related glycoproteins that are specifically associated with fibrillin-containing microfibrils. MAGP-2, unlike MAGP-1, contains an RGD motif with potential for integrin binding. To determine if the RGD sequence is active, a series of cell binding assays was performed. MAGP-2 was shown to promote the attachment and spreading of bovine nuchal ligament fibroblasts when coated onto plastic wells in molar quantities similar to those of fibronectin. In contrast, ∼10-fold more MAGP-1 was required to support comparable levels of cell adhesion. The fibroblast binding to MAGP-2 was completely inhibited if the peptide GRGDSP or the MAGP-2-specific peptide GVSGQRGDDVTTVTSET was added to the reaction medium at a 10 μm final concentration. The control peptide GRGESP had no effect on the interaction. These findings indicate that the cell interaction with MAGP-2 is an RGD-mediated event. A monoclonal antibody to human αVβ3 integrin (LM609) almost completely blocked cell attachment to MAGP-2 when added to the medium at 0.5 μg/ml, whereas two monoclonal antibodies specific for the human β1 integrin subunit, 4B4 (blocking) and QE2.E5 (activating), had no effect even at 10 μg/ml. Fetal bovine aortic smooth muscle cells, ear cartilage chondrocytes, and arterial endothelial cells and human skin fibroblasts and osteoblasts were also observed to adhere strongly to MAGP-2. In addition, each cell type was able to spread on MAGP-2 substrate, with the exception of the endothelial cells, which remained spherical after 2 h of incubation. The binding of each cell type was blocked when the anti-αVβ3 integrin antibody was included in the assay, indicating that αVβ3 integrin is the major receptor for MAGP-2 on several cell types. Thus, MAGP-2 may mediate interactions between fibrillin-containing microfibrils and cell surfaces during the development of a variety of tissues.

Immunohistochemical and Ultrastructural Localization of MP78/70 (βig-h3) in Extracellular Matrix of Developing and Mature Bovine Tissues

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, βig-h3. In the present study an antibody raised to a synthetic βig-h3 peptide was shown specifically to identify MP78/70 in purified form and in bovine tissue extracts. This is consistent with MP78/70 and βig-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.

βig-h3 Interacts Directly with Biglycan and Decorin, Promotes Collagen VI Aggregation, and Participates in Ternary Complexing with These Macromolecules

Recombinant human βig-h3 was found to bind 125I-labeled small leucine-rich proteoglycans (SLRPs), biglycan, and decorin, in co-immunoprecipitation experiments. In each instance the binding could be blocked by an excess of the unlabeled proteoglycan, confirming the specificity of the interaction. Scatchard analysis showed that biglycan bound βig-h3 more avidly than decorin with Kd values estimated as 5.88 × 10–8 and 1.02 × 10–7 m, respectively. In reciprocal blocking experiments both proteoglycans inhibited the others binding to βig-h3 indicating that they may share the same binding site or that the two binding sites are in close proximity on the βig-h3 molecule. Since βig-h3 and the SLRPs are known to be associated with the amino-terminal region of collagen VI in tissue microfibrils, the effects of including collagen VI in the incubations were investigated. Co-immunoprecipitation of 125I-labeled biglycan incubated with equimolar mixtures of βig-h3 and pepsin-collagen VI was increased 6-fold over βig-h3 alone and 3-fold over collagen VI alone. Similar increases were also observed for decorin. The findings indicate that βig-h3 participates in a ternary complex with collagen VI and SLRPs. Static light scattering techniques were used to show that βig-h3 rapidly forms very high molecular weight complexes with both native and pepsin-collagen VI, either alone or with the SLRPs. Indeed βig-h3 was shown to form a complex with collagen VI and biglycan, which appeared to be much more extensive than that formed by βig-h3 with collagen VI and decorin or those formed between the collagen and βig-h3, biglycan, or decorin alone. Biglycan core protein was shown to inhibit the extent of complexing of βig-h3 with native and pepsin-collagen VI suggesting that the glycosaminoglycan side chains of the proteoglycan were important for the formation of the large ternary complexes. Further studies showed that the direct interaction between βig-h3 and biglycan and between biglycan and collagen VI were also important for the formation of these complexes. The globular domains of collagen VI also appeared to have an influence on the interaction of the three components. Overall the results indicate that βig-h3 can differentially modulate the aggregation of collagen VI with biglycan and decorin. Thus this interplay is likely to be important in tissues such as cornea where such complexes are considered to occur.

The immunohistochemical localisation of microfibril-associated glycoprotein (MAGP) in elastic and non-elastic tissues

We have previously identified the major antigen of elastin‐associated microfibrils as a 31kD glycoprotein which we named microfibril‐associated glycoprotein or MAGP. Affinity‐purified antibodies to MAGP were shown to localise specifically to elastin‐associated microfibrils in sections of bovine foetal nuchat ligament (20). In the present paper we compare the localisation of anti‐MAGP antibodies and anti‐tropoelaslin antibodies in a range of bovine elastic and non‐elastic tissues. The results show that anti‐MAGP antibodies invariably localised to immuno‐reactive elastic fibres, wherever they occurred. Extensive additional localisation was observed in a number of tissues. This extra distribution of anti‐MAGP antibodies was found to correspond to those structures exhibiting the oxytalan histochemical staining reaction in tissues such as skin, periodontal ligament and ocular zorule. Since these oxytalan fibres have been shown to consist of 12 nm microfibrils which are morphologically similar to those of elastic fibres (and unpublished data from this laboratory confirm this conclusion), the results suggest that MAGP is a component of 12 nm microfibrils in both elastic and non‐elastic tissues. Anti‐tropoelastin antibodies did not localise to these oxytalan fibres, suggesting that tropoetastin is not a component of 12 nm microfibrils. MAGP was also detected in extracellular matrix regions of tissues such as skeletal muscle. Achilles tendon and spleen, suggesting that 12 nm microfibrils, containing one or more macromolecular constituents in common, make up an important structural system within the extracellular matrix in a wide range of elastic and non‐elastic tissues.