Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells

Abstract

N‐Glycosylation, biosynthesis and degradation of dipeptidylpeptidase IV (EC 3.4.14.5) (DPP IV) were comparatively studied in primary cultured rat hepatocytes and Morris hepatoma 7777 cells (MH 7777 cells). DPP IV had a molecular mass of 105 kDa in rat hepatocytes and of 103 kDa in MH 7777 cells as assessed by SDS/PAGE under reducing conditions. This difference in molecular mass was caused by differences in covalently attached N‐glycans. DPP IV from hepatoma cells contained a higher proportion of N‐glycans of the oligomannosidic or hybrid type and therefore migrated at a slightly lower molecular mass. In both cell types DPP IV was initially synthesized as a 97‐kDa precursor which was completely susceptible to digestion with endo‐β‐N‐acetylglucosaminidase H converting the molecular mass to 84 kDa. The precursor was processed to the mature forms of DPP IV, glycosylated with N‐glycans mainly of the complex type with a half‐life of 20–25 min. The transit of newly synthesized DPP IV to the cell surface displayed identical or very similar kinetics in both cell types with the major portion of DPP IV appearing at the cell surface after 60 min. DPP IV molecules were very slowly degraded in hepatocytes as well as in hepatoma cells with half‐lives of approximately 45 h. Inhibition of oligosaccharide processing with 1‐deoxymannojirimycin led to the formation of DPP IV molecules containing N‐glycans of the oligomannosidic type. This glycosylation variant was degraded with the same half‐life as complex‐type glycosylated DPP IV. By contrast, inhibition of N‐glycosylation with tunicamycin resulted into rapid degradation of non‐N‐glycosylated DPP IV molecules in both cell types. Non‐N‐glycosylated DPP IV could not be detected at the cell surface indicating an intracellular proteolytic process soon after biosynthesis.

Comparative study of high‐mannose‐type oligosaccharides in membrane glycoproteins of rat hepatocytes and different rat hepatoma cell lines

“…In contrast, using endo H, 30-50% of the radiolabelled N-glycans were released from the membrane glycoprotein fractions of rat hepatoma cell lines NA-MH 7777 (31.5%), MH,C,-MH 7795 (37.2%) and HTC-MH 7288c (48.0%). These results agree with structural studies on the carbohydrate chains of y-glutamyltranspeptidase from rat and human hepatoma, showing that oligomannosidic N-glycans are present only in the en-zyme of human hepatocellular carcinoma and rat AH 66 hepatoma, but that high-mannose-type oligosaccharide are absent in the enzyme of rat and human liver [12, 131. Furthermore, the data are good agreement with recent pulsekhase studies on the membrane glycoprotein dipeptidylpeptidase IV, showing that in rat hepatocytes the N-glycans of the mature form of this glycoprotein are almost completely of the complex type, whereas in Morris hepatoma cells (MH 7777) an increased proportion of high-mannose-type oligosaccharides was observed [29].…”

Endosomal Proteolysis of Glucagon at Neutral pH Generates the Bioactive Degradation Product Miniglucagon-(19–29)

“…Thus, the neutral glucagon-processing enzyme differs from the following: 1) IDE, a metalloendopeptidase of 110 kDa that generates glucagon degradation products that are structurally different from those described in the present study (9) and 2) the serine protease dipeptidyl peptidase IV, which is relatively insensitive to PMSF (55), displays a molecular mass of 105 kDa in rat hepatocytes (56), and removes the Nterminal dipeptides His 1 -Ser 2 and Gln 3 -Gly 4 from native glucagon (14). However, other members of the subtilisin-like proprotein convertase (PCs) (i.e.…”

Domain‐Specific N‐Glycosylation of the Membrane Glycoprotein Dipeptidylpeptidase IV (CD26) Influences its Subcellular Trafficking, Biological Stability, Enzyme Activity and Protein Folding

“…Although a large number of studies have been performed on a variety of glycoproteins, using glycosylation inhibitors (Elbein, 1991 ;Loch et al, 1992), the presence of nonspecific and indirect effects may interfere in the interpretation of the data. In order to investigate the function of the N-glycosylation of a single protein such as DPPIV more specifically, site-directed mutagenesis was used in the present study to abolish the addition of single oligosaccharides to different domains of DPPIV.…”

“…Following inhibition of primary N-glycosylation by tunicamycin, the biological stability of N-glycosylated DPPIV was dramatically reduced, and this protein could not be detected at the cell surface. However, inhibition of processing by l -deoxymannojirimycin led to the formation of DPPlV molecules containing N-glycans of the oligomannosidic type, but did not affect the activity of the enzyme, its stability and cell-surface transport (Loch et al, 1992). In inhibitor-treated cells, nonspecific inhibition of glycosylation of many other proteins may complicate the interpretation of the results.…”