Effects of fibrillin-1 degradation on microfibril ultrastructure.
Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.
Kuo
Chiu-Liang
CL
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.
Isogai
Zenzo
Z
Keene
Douglas R
DR
Hazeki
Noriko
N
Ono
Robert N
RN
Sengle
Gerhard
G
Bächinger
Hans Peter
HP
Sakai
Lynn Y
LY
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D017364:Collagenases / Q000502:physiology; D004548:Elasticity; D005321:Extraembryonic Membranes / Q000378:metabolism / Q000648:ultrastructure; D000071838:Fibrillin-1; D000071837:Fibrillins; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D020894:Microfibrils / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D054392:Adipokines
D017364:Collagenases; D000071838:Fibrillin-1; D000071837:Fibrillins; D019791:Guanidine; D008840:Microfilament Proteins; D010446:Peptide Fragments; C000605722:FBN1 protein, human; D054392:Adipokines