Gastric MUC5AC and MUC6 mucins were studied using polyclonal antibodies. Immunohistochemistry showed MUC5AC to originate from the surface epithelium, whereas MUC6 was produced by the glands. Mucins from the surface epithelium or glands of corpus and antrum were purified using CsCl/4M guanidinium chloride density-gradient centrifugation. MUC5AC appeared as two distinct populations at 1.4 and 1.3g/ml, whereas MUC6, which was enriched in the gland tissue, appeared at 1.45g/ml. Reactivity with antibodies against the Leb structure (where Le represents the Lewis antigen) followed the MUC5AC distribution, whereas antibodies against the Ley structure and reactivity with the GlcNAc-selective Solanum tuberosum lectin coincided with MUC6, suggesting that the two mucins are glycosylated differently. Rate-zonal centrifugation of whole mucins and reduced subunits showed that both gastric MUC5AC and MUC6 are oligomeric glycoproteins composed of disulphide-bond linked subunits and that oligomeric MUC5AC was apparently smaller than MUC6. A heterogeneous population of ‘low-density’ MUC5AC mucins, which were smaller than the ‘high-density’ ones both before and after reduction, reacted with an antibody against a variable number tandem repeat sequence within MUC5AC, suggesting that they represent precursor forms of this mucin. Following ion-exchange HPLC, both MUC5AC and MUC6 appeared as several distinct populations, probably corresponding to ‘glycoforms’ of the mucins, the most highly charged of which were found in the gland tissue.
Abbreviations used: AP, alkaline phosphatase; DFP, di-isopropyl phosphorofluoridate; DTT, dithiothreitol; HRP, horseradish peroxidase; Le, Lewis antigen; PBST, PBS containing 0.05% (v/v) Tween 20; STA, Solanum tuberosum; TBS, Tris-buffered saline; TBST, TBS containing 0.05% (v/v) Tween 20; VNTR, variable number tandem repeat.
These authors contributed equally to this work.