Characterization of a monoclonal antibody recognizing small collagenous proteins in fetal bone.
A monoclonal antibody (MBP-322) that recognizes two small collagenous, apatite-binding (SCAB) proteins associated with the mineral phase of fetal bone, has been prepared. The SCAB proteins, which are quantitatively extracted from bone with EDTA, have been shown by immunotransfer analyses to have MrS of 28K and 25K and both were selectively degraded by bacterial collagenase. Amino acid analysis of the collagenase-digested protein revealed a hypro:pro:gly ratio of approximately 0.5:1:0.7 for both proteins and indicated that one-third of the protein could have a collagen-like sequence. The SCAB proteins, unlike other collagens and collagen fragments tested bound quantitatively to hydroxylapatite in the presence of 4M guanidine hydrochloride and appear to be unique to bone. The antibody, however, was not specific for the SCAB proteins and showed comparable immunoreactivity against denatured alpha 1 chains of types I, II and III collagens and the alpha 2 chains of types I and V collagens but not type IV collagen nor native collagens I-V. The epitope was further localized to the CB6 fragment in the alpha 1(I) chain and the CB5 fragment of alpha 1(III) chain, and was present in both the TCA and TCB fragments of alpha 2(I). Despite the immunological reactivity, the properties of the SCAB proteins were not consistent with their being derived from known collagen types. Immunocytochemical staining of permeabilized bone cells with MPB-322 showed a perinuclear, punctate staining pattern in most cells with some cells showing specific nuclear staining. In non-permeabilized cells, the antibody stained various sized spherical particles, many of which were closely associated with the cell surface. Immunoblots of cell proteins revealed a number of immunoreactive proteins sensitive to collagenase digestion including two proteins with MrS similar to the SCAB proteins. The MBP-322 antibody appears useful for identifying sequence homology in various collagens, and for recognizing denatured collagen and specific collagen fragments in tissues, as well as being important for the further characterization of the SCAB proteins.
Kuwata
F
F
Maeno
M
M
Yao
K L
KL
Domenicucci
C
C
Goldberg
H A
HA
Wasi
S
S
Aubin
J E
JE
Sodek
J
J
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D001842:Bone and Bones / Q000166:cytology / Q000196:embryology*; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005333:Fetus; D005455:Fluorescent Antibody Technique; D008970:Molecular Weight; D013552:Swine
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003094:Collagen