The isolated MUC5AC gene product from human ocular mucin displays intramolecular conformational heterogeneity.
Atomic force microscopy (AFM) has been used to show that human ocular mucins contain at least three distinct polymer conformations, separable by isopycnic density gradient centrifugation. In this work we have used affinity purification against the anti(mucin peptide core) monoclonal antibody 45M1 to isolate MUC5AC gene products, a major component of human ocular mucins. AFM images confirm that the affinity-purified polymers adopt distinct conformations that coidentify with two of those observed in the parent population, and further reveal that these two different conformations can be present within the same polymer. AFM images of the complexes formed after incubation of 45M1 with the parent sample reveal different rates of binding to the two MUC5AC polymer types. The variability of gene products within a mucin population was revealed by analyzing the height distributions along the polymer contour and periodicities in distances between occupied antibody binding sites. AFM analysis of mucin polymers at the single molecule level provides new information about the genetic origins of individual polymers and the contributions of glycosylation to the physicochemical properties of mucins, which can be correlated with information obtained from biochemistry, antibody binding assays, and molecular biology techniques.
Round
Andrew N
AN
H.H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol BS8 1TL, UK. andy.round@bristol.ac.uk
McMaster
Terence J
TJ
Miles
Mervyn J
MJ
Corfield
Anthony P
AP
Berry
Monica
M
D000911:Antibodies, Monoclonal / Q000378:metabolism; D003228:Conjunctiva / Q000737:chemistry; D005123:Eye / Q000737:chemistry*; D006801:Humans; D055271:Mucin 5AC; D009077:Mucins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D011108:Polymers