Evidence of in situ stability of the type IV collagen triple helix in human inflammatory bowel disease using a denaturation specific epitope antibody.
A peptide specific antibody (AH1OW1) was raised against an epitope, AH10 (aa 449-463), of the alpha1(IV) chain adjacent to a cleavage site for matrix metalloproteinases (MMP)-2 and -9 within the triple helix of type IV collagen. The antibody only reacted with denatured and reduced preparations of type IV collagen, or with pepsin isolated type IV collagen digested with MMP-2 and MMP-9. The specificity of this antibody for the denatured triple helix was demonstrated by the lack of staining with pre-immune antibody and by pre-incubation of AH1OW1 antibody with excess AH10 peptide epitope. The AH1OWI antibody was used to detect whether proteolysis of type IV collagen occurs in ulcerative colitis, an inflammatory bowel condition often characterised by a large influx of granulocytes and macrophages and an associated tissue destruction. However, no evidence of in situ proteolysis of the basement membrane type IV collagen was observed. Only in the most actively inflamed mucosa was staining with AH1OW1 antibody observed in the mucosal connective tissue. Digestion of frozen sections of bowel with MMP-1, MMP-2, MMP-3 and MMP-9 did not result in the exposure of the AH10 epitope. These data demonstrate the stability of intact type IV collagen and indicate that susceptibility of alpha1(IV) chain to digestion with MMP-2 and MMP-9 may require other proteolytic/denaturing events in the molecule.
Wheatcroft
A C
AC
Division of Human Metabolism, Royal Hallamshire Hospital, Sheffield, UK.
Hollander
A P
AP
Croucher
L J
LJ
Jones
A
A
Taylor
C J
CJ
Evans
G S
GS
D000328:Adult; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D000918:Antibody Specificity; D015153:Blotting, Western; D002648:Child; D002908:Chronic Disease; D003093:Colitis, Ulcerative / Q000378:metabolism; D003094:Collagen / Q000276:immunology / Q000378:metabolism*; D003106:Colon / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D007150:Immunohistochemistry; D015212:Inflammatory Bowel Diseases / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D011489:Protein Denaturation; D011817:Rabbits
D000906:Antibodies; D000939:Epitopes; D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9