Determination of the epitope for the inhibitory monoclonal antibody 5-B6 on the catalytic subunit of gastric Mg2+-dependent H+-transporting and K+-stimulated ATPase

Abstract

The monoclonal antibody 5-B6, directed against the alpha-subunit of pig gastric H+,K(+)-ATPase (Mg(2+)-dependent H(+)-transporting and K(+)-stimulated ATPase), was shown to be a potent inhibitor of the K(+)-ATPase activity, thereby binding to the cytoplasmic side of the alpha-subunit of the enzyme [Van Uem, Peters & De Pont (1990) Biochim. Biophys. Acta 1023, 56-62]. In order to define the epitope for 5-B6 on pig gastric H+,K(+)-ATPase more precisely, the alpha-subunit of the enzyme was subjected to limited proteolysis followed by chemical cleavage. Restricted proteolysis with papain followed by sequence analysis yielded an immunoreactive fragment of 27 kDa beginning at Ser379. This fragment was water-soluble and possessed the fluorescein isothiocyanate-reaction site. Limited tryptic digestion in the presence of K+ gave rise to an immunoreactive 56 kDa fragment beginning at Ile456, thus restricting the location of the epitope from Ile456 to the C-terminal end of the 27 kDa fragment (around residue 620). Further degradation of the 27 kDa fragment by means of formic acid cleavage at Asp-Pro bonds resulted initially in the formation of two non-immunoreactive fragments of 17 kDa and 11 kDa, indicating that the epitope for 5-B6 has to be localized around the chemical cleavage sites Asp507 and/or Asp510. Comparison of the primary structure of the alpha-subunits of gastric H+,K(+)-ATPase and non-immunoreactive rat kidney Na+,K(+)-ATPase shows almost no similarity for the sequence containing these formic acid cleavage sites (Thr504-Leu-Glu-Asp-Pro-Arg-Asp-Pro-Arg512), whereas the adjacent sequences are nearly 100% identical. These findings strongly suggest that the epitope for 5-B6 includes (part of) this sequence.

Characterization of a Protease-resistant Domain of the Cytosolic Portion of Sarcoplasmic Reticulum Ca2+-ATPase

“…We found no evidence for a particular sensitivity of the Arg 505 -Ala 506 peptidic bond to proteases other than trypsin; thus, the high sensitivity to trypsin of this bond is probably due to a particular conformation of the Arg 505 -Ala 506 peptidic bond, favorable for trypsinolysis, rather than to a strategic location of this bond at the boundary between two distinct domains of Ca 2ϩ -ATPase. It is remarkable that proteolysis fragments similar to our p29/30 fragments have in fact been already described for Na ϩ ,K ϩ -ATPase and H ϩ ,K ϩ -ATPase, after treatment with either trypsin (42) or other proteases (43)(44)(45)(46).…”

Constitutive activation of gastric H+,K+-ATPase by a single mutation

“…There are several indications that binding of K + to its binding pocket in the membrane has an effect on the conformation of the large intracellular loop between the transmembrane segments four and five, in which K + binding and phosphorylation occur. A readily accessible tryptic digestion site at Lys668 is no longer available in the presence of K + , whereas a cleavage site at Arg455 is exposed (Helmich‐de Jong et al ., 1987; Munson et al ., 1991; Van Uem et al ., 1991). Fluorescence by fluorescein isothiocyanate (Jackson et al ., 1983) and eosin (Helmich‐de Jong et al ., 1986), both of which agents interact with the intracellular loop, is affected by K + .…”

“…Fluorescence by fluorescein isothiocyanate (Jackson et al ., 1983) and eosin (Helmich‐de Jong et al ., 1986), both of which agents interact with the intracellular loop, is affected by K + . The binding of the monoclonal antibody 5B6 to this loop is also affected by K + (Van Uem et al ., 1990, 1991). The conformational change in the loop could have an effect on the accessibility of the aspartyl–phosphate bond for water.…”

Role of Negatively Charged Residues in the Fifth and Sixth Transmembrane Domains of the Catalytic Subunit of Gastric H+,K+-ATPase

“…The amount of H ϩ ,K ϩ -ATPase protein expressed was measured using an enzyme-linked immunosorbent assay (40), based on specific binding of the produced protein to the monoclonal antibody 5B6 (41), which recognizes an epitope in the intracellular loop between the fourth and fifth transmembrane domain of the catalytic subunit of pig gastric H ϩ ,K ϩ -ATPase (42). By comparing the amount of immunoreactive protein with that of the pig enzyme, the amount of baculovirus-produced (mutated) H ϩ ,K ϩ -ATPase could be quantitated.…”