Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing.

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.

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Characterization of muscle epimysium, perimysium and endomysium collagens

Biochemical Journal ◽

10.1042/bj2191017 ◽

1984 ◽

Vol 219 (3) ◽

pp. 1017-1026 ◽

Cited By ~ 141

Author(s):

N Light ◽

A E Champion

Keyword(s):

Connective Tissue ◽

Sodium Dodecyl Sulphate ◽

Type Iv Collagen ◽

Sodium Dodecyl ◽

Basement Membranes ◽

Type Iii Collagen ◽

Type I ◽

Type V Collagen ◽

Type Iv ◽

Type V

In the past it has been proven difficult to separate and characterize collagen from muscle because of its relative paucity in this tissue. The present report presents a comprehensive methodology, combining methods previously described by McCollester [(1962) Biochim. Biophys. Acta 57, 427-437] and Laurent, Cockerill, McAnulty & Hastings [(1981) Anal. Biochem. 113, 301-312], in which the three major tracts of muscle connective tissue, the epimysium, perimysium and endomysium, may be prepared and separated from the bulk of muscle protein. Connective tissue thus prepared may be washed with salt and treated with pepsin to liberate soluble native collagen, or can be washed with sodium dodecyl sulphate to produce a very clean insoluble collagenous product. This latter type of preparation may be used for quantification of the ratio of the major genetic forms of collagen or for measurement of reducible cross-link content to give reproducible results. It was shown that both the epimysium and perimysium contain type I collagen as the major component and type III collagen as a minor component; perimysium also contained traces of type V collagen. The endomysium, the sheaths of individual muscle fibres, was shown to contain both type I and type III collagen as major components. Type V collagen was also present in small amounts, and type IV collagen, the collagenous component of basement membranes, was purified from endomysial preparations. This is the first biochemical demonstration of the presence of type IV collagen in muscle endomysium. The preparation was shown to be very similar to other type IV collagens from other basement membranes on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and was indistinguishable from EHS sarcoma collagen and placenta type IV collagen in the electron microscope after rotary shadowing.

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Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen.

The Journal of Cell Biology ◽

10.1083/jcb.95.2.641 ◽

1982 ◽

Vol 95 (2) ◽

pp. 641-647 ◽

Cited By ~ 52

Author(s):

J M Fitch ◽

E Gibney ◽

R D Sanderson ◽

R Mayne ◽

T F Linsenmayer

Keyword(s):

Monoclonal Antibody ◽

Basement Membrane ◽

Type Iv Collagen ◽

Helical Structure ◽

Immunohistochemical Localization ◽

Basement Membranes ◽

Optical Rotatory Dispersion ◽

Collagen Molecule ◽

Type Iv ◽

Triple Helical Domain

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.

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Isolation and native characterization of cysteine-rich collagens from bovine placental tissues and uterus and their relationship to types IV and V collagens

Bioscience Reports ◽

10.1007/bf01115247 ◽

1982 ◽

Vol 2 (7) ◽

pp. 493-502 ◽

Cited By ~ 41

Author(s):

M. Z. Abedin ◽

Shirley Ayad ◽

Jacqueline B. Weiss

Keyword(s):

Type Iv Collagen ◽

Deae Cellulose ◽

Collagen Types ◽

Type V Collagen ◽

Type Iv ◽

Probable Relationship ◽

Simplified Procedure ◽

Type V

A simplified procedure for the fractionation and purification of different collagen types from various tissues is described which is particularly efficient in separating type-V from type-IV collagen, and highmol.-wt. (HMW) aggregates from 7 S collagen. Uterus and maternal villi contain 2 forms of type-V collagen −{α1(V)}2α2(V) and {α1(V)2α2(V)α3(V)}–which have been separated on DEAE-cellulose. Uterus however appears to be the richest source of both HMW aggregates and the {α1(V)2α2(V)α3(V)} collagen, and a probable relationship between these collagens is discussed.

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Cell type specific recognition of the reconstituted aggregates from isolated type I collagen, type IV collagen, and type V collagen

Journal of Chemical Sciences ◽

10.1007/bf02869906 ◽

1999 ◽

Vol 111 (1) ◽

pp. 171-177

Author(s):

Toshihiko Hayashi ◽

Kazunori Mizuno ◽

Motohiro Hirose ◽

Koichi Nakazato ◽

Eijiro Adachi ◽

...

Keyword(s):

Type Iv Collagen ◽

Type I Collagen ◽

Collagen Type ◽

Collagen Type Iv ◽

Type I ◽

Cell Type ◽

Type V Collagen ◽

Type Iv ◽

Cell Type Specific ◽

Type V

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Structural studies of human basement-membrane collagen with the use of a monoclonal antibody

Biochemical Journal ◽

10.1042/bj2270217 ◽

1985 ◽

Vol 227 (1) ◽

pp. 217-222 ◽

Cited By ~ 24

Author(s):

H Dieringer ◽

D W Hollister ◽

R W Glanville ◽

L Y Sakai ◽

K Kühn

Keyword(s):

Monoclonal Antibody ◽

Basement Membrane ◽

Type Iv Collagen ◽

Electron Microscopic Observation ◽

Electron Microscopic ◽

Type Iv ◽

Structural Probe ◽

Basement Membrane Collagen ◽

Antigen Antibody ◽

Membrane Collagen

A monoclonal antibody monospecific for human type IV collagen was used as a structural probe to examine aspects of the macromolecular organization of basement-membrane collagen. Electron-microscopic observation of rotary-shadowed antigen-antibody complexes demonstrated a unique binding site for the antibody 55 +/- 6 nm distant from the 7S cross-linking region of tetrameric type IV collagen. This observation allowed a series of studies that showed: (1) the localization of an intramolecular disulphide bridge within the helical domain of the molecule, (2) the alignment of major peptic-digest fragments of the alpha 1 (IV) chain, and (3) confirmation of the postulated antiparallel arrangement of individual molecules within type IV collagen tetramers.

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ChemInform Abstract: Cell Type Specific Recognition of the Reconstituted Aggregates from Isolated Type I Collagen, Type IV Collagen, and Type V Collagen

ChemInform ◽

10.1002/chin.199930309 ◽

2010 ◽

Vol 30 (30) ◽

pp. no-no

Author(s):

Toshihiko Hayashi ◽

Kazunori Mizuno ◽

Motohiro Hirose ◽

Koichi Nakazato ◽

Eijiro Adachi ◽

...

Keyword(s):

Type Iv Collagen ◽

Type I Collagen ◽

Collagen Type ◽

Collagen Type Iv ◽

Type I ◽

Cell Type ◽

Type V Collagen ◽

Type Iv ◽

Cell Type Specific ◽

Type V

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Collagen type I and type V are present in the same fibril in the avian corneal stroma.

The Journal of Cell Biology ◽

10.1083/jcb.106.3.999 ◽

1988 ◽

Vol 106 (3) ◽

pp. 999-1008 ◽

Cited By ~ 265

Author(s):

D E Birk ◽

J M Fitch ◽

J P Babiarz ◽

T F Linsenmayer

Keyword(s):

Monoclonal Antibodies ◽

Collagen Type ◽

Corneal Stroma ◽

Collagen Type I ◽

Collagen Molecule ◽

Type I ◽

Collagen Types ◽

Type V Collagen ◽

Fibril Structure ◽

Type V

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.

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The use of 1nm silver enhanced gold probes for the detection of skin antigens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162077 ◽

1990 ◽

Vol 48 (3) ◽

pp. 902-903

Author(s):

J.P Cassella ◽

H. Shimizu ◽

A. Ishida-Yamamoto ◽

R.A.J. Eady

Keyword(s):

Type Iv Collagen ◽

Freeze Substitution ◽

Collagen Type Iv ◽

Electron Microscopic ◽

Normal Human Skin ◽

Type Iv ◽

Type Vii Collagen ◽

Keratin Filaments ◽

Normal Human ◽

Phosphate Buffered Saline

1nm colloidal gold with silver enhancement has been used in conjunction with a low-temperature post-embedding (post-E) technique for the demonstration of skin antigens at both the light microscopic (LM) and electron microscopic (EM) levels.Keratin filaments and basement membrane zone (BMZ) associated antigens in normal human skin (NHS) were immunolabelled using antibodies against keratin 14, 10, and 1, the carboxy-terminus and collagenous portion of type VII collagen, type IV collagen and bullous pemphigoid antigen (BP-Ag).Fresh samples of NHS were cryoprotected in 15% glycerol, cryofixed in propane at -190°C, subjected to freeze substitution in methanol at -80°C and embedded in Lowicryl K11M at -60°C. Polymerisation of the resin was initiated under UVR at - 60°C for 48 hours and continued at room temperature for a further 48 hours. Semith in sections were air dried onto slides coated with 3-aminopropyltriethoxysilane. The following immunolabelling protocol was adopted: Primary antibody was applied for 2 hours at 37°C or overnight at 4°C. Following washing in Dulbecco’s phosphate buffered saline (PBSA) a biotinylated secondary antibody was applied for 2 hours at 37°C. The sections were further washed in PBSA and 1nm gold avidin was applied. Sections were finally washed in PBSA and silver enhanced.

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