Sialoforms of dipeptidylpeptidase IV from rat kidney and liver

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and,endopeptidase found in the cell surface membrane of many tissues, was employed as a mode l membrane glycoprotein to study the expression of sialoforms on cell surfa ce glycoproteins. Native, enzymatically active DPP IV was purified from pla sma membranes of kidney and liver by lectin affinity chromatography in conj unction with crown ether anion exchange chromatography, The enzyme was grad ient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide ge l electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysi s of the purified DPP IV by isoelectric focusing (IEF) showed that it consi sts of several polypeptides of different isoelectric points (IP) ranging fr om 5.5 to 7.0, In vitro-desialylation of the enzyme and subsequent isoelect ric focusing revealed that the differences in isoelectric points mere due t o differences in the degree of sialylation. Differences in the degree of si alylation between the fractions were also demonstrated by SDS-PAGE under no nreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration ve locity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro-d esialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzy me was predominantly sialylated via alpha 2,6-linkage, as shown by lectin a ffinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amur ensis agglutinin (MAA), These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each othe r by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.