TY - JOUR
T1 - Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies
AU - Hisae, Hori
AU - Keene, Douglas R.
AU - Sakai, Lynn Y.
AU - Wirtz, Mary K.
AU - Bächinger, Hans Peter
AU - Godfrey, Maurice
AU - Hollister, David W.
N1 - Funding Information:
*Supported by grants from the Shriners Hospitals of North America, and by a grant from NIAMSD (AR37272). Electron Microscopy Facilities were provided in part by the Fred Meyer Charitable Trust and the R. Blame Bramble Medical Research Foundations. TPresent address: Department of Tissue Physiology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan. (Author to whom correspondence should be addressed.) JPresent address: Department of Pediatrics, Hollister Connec-tive Tissue Disease Research Laboratory, University of Nebraska Medical Center, Omaha, NE 68198-5430, U.S.A. §Deceased. Abbreviations: BSA, bovine serum albumin; CB, cyanogen bromide cleaved; ELISA, enzyme-linked immunosor~nt assay; HAT, hypoxanthine-aminopterin-thymidine; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; TEM, transmission electron microscopy; T,, meiting temperature.
PY - 1992/6
Y1 - 1992/6
N2 - Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the tN-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller tC-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4°C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.
AB - Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the tN-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller tC-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4°C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.
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U2 - 10.1016/0161-5890(92)90186-2
DO - 10.1016/0161-5890(92)90186-2
M3 - Article
C2 - 1376414
AN - SCOPUS:0026711158
SN - 0161-5890
VL - 29
SP - 759
EP - 770
JO - Molecular Immunology
JF - Molecular Immunology
IS - 6
ER -