Glycobiology, 1999, Vol. 9, No. 12 1295-1305
© 1999 Oxford University Press
Sialoforms of dipeptidylpeptidaseIV from rat kidney and liver
Institut für Molekularbiologie und Biochemie derFreien Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem,Germany and 4Institut fürKlinische Chemie und Biochemie, Virchow-Klinikum, Medizinische Fakultätder Humboldt Universität zu Berlin, Augustenburger Platz1, D-13353 Berlin-Wedding, Germany
Dipeptidylpeptidase IV (DPP IV, CD26), a serine-typeexo- and endopeptidase found in the cell surface membrane of manytissues, was employed as a model membrane glycoprotein to studythe expression of sialoforms on cell surface glycoproteins. Native,enzymatically active DPP IV was purified from plasma membranes ofkidney and liver by lectin affinity chromatography in conjunctionwith crown ether anion exchange chromatography. The enzyme was gradient-elutedin continuous fractions, all showing a single polypeptide band ofabout 100 kDa when separated by sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE) under reducing, denaturingconditions. Analysis of the purified DPP IV by isoelectric focusing (IEF)showed that it consists of several polypeptides of different isoelectricpoints (IP) ranging from 5.5 to 7.0. In vitro-desialylationof the enzyme and subsequent isoelectric focusing revealed thatthe differences in isoelectric points were due to differences inthe degree of sialylation. Differences in the degree of sialylationbetween the fractions were also demonstrated by SDS–PAGEunder nonreducing and nondenaturing conditions. Increased sialylationof the enzyme as demonstrated by isoelectric focusing resulted in increasedmigration velocity in nonreducing and nondenaturing SDS–polyacrylamidegels. In vitro-desialylation of the enzyme andits resialylation confirmed that sialylation was responsible forthis extraordinary migration behavior. The native enzyme was predominantlysialylated via 
2,6-linkage, as shownby lectin affinity blotting employing Sambucus nigra agglutinin(SNA) and Maackia amurensis agglutinin (MAA). Thesefindings demonstrate that a distinct membrane glycoprotein may existin various sialoforms, distinguished from each other by a differentnumber of sialic acid residues. Moreover, these sialoforms can be individuallypurified by crown ether anion exchange chromatography.
a Presentaddress: Deutsches Krebsforschungszentrum, Abteilung Molekulare Toxikologie,Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
b Present address:Institut für Klinische Chemie und Pathobiochemie, UniversitätsklinikumBenjamin-Franklin, Fachbereich Humanmedizin der Freien UniversitätBerlin, Hindenburgdamm 30, D-12200 Berlin, Germany