Direct apical sorting of rat liver dipeptidylpeptidase IV expressed in Madin-Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, apical and basolateral membrane proteins are segregated from each other in the trans-Golgi network (TGN) and are transported to the appropriate membrane domain via separate vesicle populations. In hepatocytes, however, all plasma membrane proteins are delivered basolaterally. Apical proteins are then selectively retrieved and reach the apical surface by transcytosis. The sorting of apical proteins in different cell types may be the result of differences in the cellular sorting machinery, or alternatively, due to expression of cell-specific sorting signals on the proteins themselves. To test this directly, we have stably expressed cDNA encoding an apical protein from rat liver, dipeptidylpeptidase IV (DPPIV), in MDCK cells. We found that approximately 90% of the exogenous DPPIV is expressed on the apical cell surface at steady state. Furthermore, we demonstrate that this distribution is primarily due to vectorial transport from the TGN to the apical plasma membrane. The small pool of mis-sorted DPPIV that appears basolaterally is slowly endocytosed (t1/2 approximately 60 min) and is subsequently transcytosed. These data are consistent with the notion that both hepatocytes and MDCK cells are capable of correctly sorting rat liver DPPIV, but that this sorting occurs at different sites in the two cell types.     

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 model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.     

Reaction of dipeptidylpeptidase IV with substrate-analogous azapeptides.

The reaction of dipeptidyl peptidase IV (EC 3.4.14.5.) with azapeptide substrates containing azaalanine or azaproline in the P1-position was investigated. Accumulation of a fairly stable acyl-enzyme could be shown for ester substrates. Ala-AzaPro-pNA is a very poor substrate of DP IV and does not accumulate an acyl-enzyme. DP IV does not react with active-site titrants for trypsin-like serine proteases.     

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

Identification of cell surface dipeptidylpeptidase IV in human fibroblasts.

An antigen with dipeptidylpeptidase IV activity was identified at the surface of normal human fibroblasts. Hydrophobic interaction electrophoresis in phenyl-Sepharose revealed that the enzyme contained a hydrophobic domain, while lactoperoxidase-catalysed iodination with 125I of living cells indicated that the protein was located at the cell surface. Crossed immunoelectrophoresis with specific antibodies of acid-extracted or papain-treated cells showed a shift of the dipeptidylpeptidase IV peak to a faster mobility. The molecular properties of the fibroblast enzyme were clearly different from those described for dipeptidylpeptidase IV from other tissues and species. Fibroblast dipeptidylpeptidase IV contained two different disulphide-linked subunits, of apparent Mr values 125000 and 135000 (denatured and reduced). In gel filtration, an Mr of about 400000 was observed for the unreduced molecule. The enzymic properties of fibroblast dipeptidylpeptidase IV were very similar to those of the well-characterized pig kidney enzyme. Activity towards glycyl-L-prolyl-beta-naphthylamide was inhibited 50% by 0.023 mM-di-isopropylphosphorofluoridate. L-Alanyl-L-alanyl-beta-naphthylamide was hydrolysed ten times more slowly than glycyl-L-prolyl-beta-naphthylamide.     

Tight junctions and apical/basolateral polarity

Summary Physiological studies led to devise models of epithelial cells in which the membrane does not have its molecules distributed homogeneously, but polarized towards the apical or towards the basolateral regions. For a while, it was assumed that the TJ, acting as a fence between the two regions, would be responsible for this asymmetry. However, today the information available indicates not only that polarization may proceed independently of the TJ, but that this structure itself may attain its precise location due to a polarization process. Nevertheless, TJs may play a role in restricting to the apical or to the basolateral region those molecules that are free to diffuse in the plane of the cell membrane (e.g., lipids and protein that are not attached to cytoplasmic or extracellular structures).     

Peptidases II. Localization of dipeptidylpeptidase IV (DPP IV). Histochemical and biochemical study]

Fresh frozen, unfixed, chloroforme-acetone treated or freeze-dried cryostat sections or sections from aldehyde-fixed blocks of tissue were tried for the histochemical investigation of dipeptidylpeptidase IV (DPP IV) with L-glycyl-L-prolyl(gly-pro)-naphthylamides as substrates and stable or unstable diazonium salts for simultaneous coupling and various buffers, pH 5--7.5 in rats, mice, guinea-pigs, cats, rabbits, hamsters and human enterobiopsies. The best results are obtained with 1.7--3.4 mM gly-pro-4-methoxy-2-naphthylamide and 1 mg Fast Blue B/ml or (with some limitations) 0.025 ml hexazotized new fuchsine/ml in 0.1 M cacodylate or phosphate buffer, pH 7.5 and unfixed sections for the demonstration of the total activity of DPP IV and freeze-dried celloidin-mounted cryostat sections for the precise localization of the enzyme or the detection of lysosomes, Golgi apparatus and secretion granules sections from aldehyde fixed tissue blocks are only suitable to study the lysosomal hydrolysis of gly-pro-naphthylamides between pH 5 and 7 when hexazotized p-rosaniline or new fuchsine are employed. DPP IV is firmly bound to strutures and shows species- and organ-dependent differences. In general, the enzyme occurs in the capillary endothelium, sinusoidal cells, perineurium, epithelial cells of intercalated and striated ducts, microvillous zone of intestinal crypts and villi, uterus, Fallopian tubes, ductus epididymis and proximal renal tubules, hepatocyte and lymphocyte membrane, plasmalemma of pseudostratified and transient epithelia and in the capsules and interstitium of many organs. These sites of activity can be completely inhibited by diisopropyl fluorophosphate and partially by Pb2+; Mg2+, Mn2+, Co2+ EDTA are without any influence. Phenantrolin may activate DPP IV. The biochemical assay works with 10 mM gly-pro-2-naphthylamide in 0.1 M cacodylate buffer, pH 7; the enzyme activity is determined fluorometrically in guinea-pig and rat organs; the data confirm and enlarge the species- and organ-dependent differences revealed by histochemistry. Compared with other dipeptide as well as tripeptide and amino acid naphthylamides the results obtained for DPP IV suggest a peptidylpeptidase which seems to be involved in other metabolic processes beside the degradation of collagen.     

Changes of dipeptidylpeptidase IV as a membrane marker of lymphocytes in acute and chronic liver diseases--biochemical and cytochemical investigations.

Lymphocytic dipeptidylpeptidase IV (DPP-IV, E.C. 3.4.14.5) is described as a marker enzyme of immunostimulant T-lymphocytes as well as functional characteristic of interleukin-2-producing cells. Cytochemical staining of DPP-IV positive lymphocytes and measurements of DPP-IV activity in mononuclear cells and in sera of patients suffering from different kinds of liver diseases were performed to evaluate the average activities in positive cells. The results demonstrated that this serine exopeptidase exhibits extremely low activity in autoimmune chronic hepatopathies. On the contrary, hepatitis-B-associated liver diseases were connected with markedly increased values. Furthermore, significant differences of DPP-IV activity were found in different kinds of acute and chronic liver diseases. These findings are discussed in connection with the participation of dipeptidylpeptidase IV in impaired immunoregulation of the altered liver.     

Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information.

An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.     

Rat liver insulin mediator which stimulates pyruvate dehydrogenase phosphate contains galactosamine and D-chiroinositol

It has been established that insulin treatment of cells, isolated plasma membranes, or whole animals leads to the generation of low molecular weight mediators which serve as intermediates in the signalling pathway. At least two distinct classes of mediator have been described, based on differences in apparent molecular weight, isoelectric point and biological activity (Cheng, K., and Larner, J. (1985) Ann. Rev. Physiol. 45, 407-424). Recently, Saltiel's (Saltiel, A.R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5793-5797) and Mato's (Mato, J.M., Kelly, K.L., Abler, A., and Jarett, L. (1987) J. Biol. Chem. 262, 2131-2137) laboratories have described an insulin "modulator" which was apparently derived from glycosylphosphoinositol linker, similar to those known to anchor proteins to the external surface of the cell membrane (Low, M.G. (1987) Bioch. J. 244, 1-13). In this paper, we report that highly purified preparations of the insulin mediator which stimulates pyruvate dehydrogenase phosphatase contain mannose, galactosamine, and D-chiroinositol. These determinations are based upon analyses using paper chromatography and gas chromatography/mass spectroscopy. Nitrous acid deamination of the mediator resulted in release of inositol phosphate, indicating that the galactosamine and D-chiroinositol are linked. Although the presence of chiroinositol in modulator from H35 hepatoma cells has been recently reported (Mato, J.M., Kelly, K.L., Abler, A., Jarett, L., Corkey, B.E., Cashel, J.A., and Zopf, D. (1987) Bioch. Biophys. Res. Comm. 146, 764-770), the optical identity of the inositol remained unknown until the present report. Likewise, the presence of galactosamine rather than glucosamine in insulin mediator is a novel finding. These findings, coupled with those of Saltiel and Mato's groups, provide clear evidence for the existence of multiple forms of insulin mediators. Additionally, the results presented here afford further confirmation for the formation of insulin mediators from glycosyl-phosphoinositol linkers.     

The NH2-terminal domain of Golgin-160 contains both Golgi and nuclear targeting information

Golgin-160 is a member of the golgin family of Golgi-localized membrane proteins. The COOH-terminal two-thirds of golgin-160 is predicted to form a coiled-coil, with an NH(2)-terminal "head" domain. To identify the Golgi targeting information in golgin-160, full-length and deletion constructs tagged with green fluorescent protein were generated. The head domain alone was targeted to the Golgi complex in the absence of assembly with endogenous golgin-160. Further truncations from both ends of the head domain narrowed the Golgi targeting information to 85 amino acids between residues 172 and 257. Surprisingly, certain truncations of the head domain also specifically accumulated in the nucleus. Both a nuclear localization signal (masked in the full-length protein) and information for nuclear retention contributed to the nuclear localization of these truncations. Because the golgin-160 head is cleaved by caspases during apoptosis, we examined the localization of epitope-tagged proteins corresponding to all potential caspase cleavage fragments. Our data suggest that three of six fragments could be targeted to the nucleus, provided that they are released from Golgi membranes after cleavage. The finding that both Golgi and nuclear targeting information is present in the same region of golgin-160 suggests that this protein may have more than one function.     

The cytoplasmic/transmembrane domain of dipeptidyl peptidase IV, a type II glycoprotein, contains an apical targeting signal that does not specifically interact with lipid rafts

We investigated the signals involved in the apical targeting of dipeptidyl peptidase IV (DPP IV/CD26), an archetypal type II transmembrane glycoprotein. A secretory construct, corresponding to the DPP IV ectodomain, was first stably expressed in both the enterocytic-like cell line Caco-2 and the epithelial kidney MDCK cells. Most of the secretory form of the protein was delivered apically in MDCK cells, whereas secretion was 60% basolateral in Caco-2 cells, indicating that DPP IV ectodomain targeting is cell-type-dependent. A chimera (CTM-GFP) containing only the cytoplasmic and transmembrane domains of mouse DPP IV plus the green fluorescent protein was then studied. In both cell lines, this chimera was preferentially expressed at the apical membrane. By contrast, a secretory form of GFP was randomly secreted, indicating that GFP by itself does not contain cryptic targeting information. Comparison of the sequence of the transmembrane domain of DPP IV and several other apically targeted proteins does not show any consensus, suggesting that the apical targeting signal may be conformational. Neither the DPP IV nor the CTM-GFP chimera was enriched in lipid rafts. Together these results indicate that, besides the well-known raft-dependent apical targeting pathway, the fate of the CTM domain of DPP IV may reveal a new raft-independent apical pathway.     

The COOH-terminal tail of the GAT-2 GABA transporter contains a novel motif that plays a role in basolateral targeting

The ability of polarized epithelia to perform vectorial transport depends on the asymmetrical distribution of transmembrane proteins among their plasma membrane domains. The establishment and maintenance of these polar distributions relies on molecular signals embedded in the proteins themselves and the interpretation of these signals by cellular sorting machinery. Using Madin-Darby canine kidney (MDCK) cells as an in vitro model of polarized epithelia, our laboratory has previously shown that the COOH-terminal cytoplasmic 22 amino acids of the GAT-2 isoform of the -amino butyric acid (GABA) transporter are necessary for its basolateral distribution. We demonstrate that the COOH-terminal tail of the transporter can function as an autonomous basolateral distribution signal, independently of the rest of the transporter. We find that the three-amino acid PDZ domain-interacting motif at the COOH-terminus of GAT-2 is not necessary for its basolateral distribution. Instead, the more proximal seven amino acids are necessary both for targeting and for steady-state distribution. Because this sequence resembles no other known basolateral sorting information, we conclude that these seven amino acids contain a novel basolateral targeting and distribution motif. GABA transporter; traffic; sorting signal; targeting signal     

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells

Overexpressionof the epidermal growth factor receptors (EGFR) in polarized kidneyepithelial cells caused them to appear in high numbers at both thebasolateral and apical cell surfaces. We utilized these cells to lookfor differences in the regulation and signaling of apical vs.basolateral EGFR. Apical and basolateral EGFR were biologically activeand mediated EGF-induced cell proliferation to similar degrees.Receptor downregulation and endocytosis were less efficient at theapical surface, resulting in prolonged EGF-induced tyrosine kinaseactivity at the apical cell membrane. Tyrosine phosphorylation of EGFRsubstrates known to mediate cell proliferation, Src-homologous andcollagen protein (SHC), extracellularly regulated kinase 1 (ERK1), andERK2 could be induced similarly by activation of apical or basolateralEGFR. Focal adhesion kinase was tyrosine phosphorylated more bybasolateral than by apical EGFR; however, -catenin was tyrosinephosphorylated to a much greater degree following the activation ofmislocalized apical EGFR. Thus EGFR regulation and EGFR-mediatedphosphorylation of certain substrates differ at the apical andbasolateral cell membrane domains. This suggests that EGFRmislocalization could result in abnormal signal transduction andaberrant cell behavior.

     

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

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-beta-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.     

Rat liver contains age-regulated cytosolic 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn).

Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid), a unique deaminated member of the sialic acid family, has emerged as a new building block of glycoconjugates from a wide variety of organisms, ranging from bacteria to mammals. In particular, the presence of Kdn has been demonstrated in different rat organs and tissues, but not in liver. Here we report on the detection and quantitation of Kdn in rat liver and on its variations with postnatal development and aging. We have previously established the optimal conditions for derivatization of Kdn with 1,2-diamino-4, 5-methylene-dioxybenzene (DMB), and detection by reverse-phase HPLC. Analysis of whole liver homogenates and different subcellular fractions reveals that Kdn is fundamentally present in the cytosolic fraction as nucleotide precursor. The expression of Kdn, Neu5Gc, and Neu5Ac changes unevenly with age. While the content of Neu5Ac, the major species, and Neu5Gc decreases to a different extent from newborn to old animals, Kdn content decreases from newborn to trace amounts in adult rats and increases again with aging. Thus, expression of Kdn, Neu5Gc, and Neu5Ac appears to be independently regulated.     

Alkaline phosphatase and dipeptidylpeptidase IV staining of tissue components of skeletal muscle: a comparative study.

A combined alkaline phosphatase (AP) and dipeptidlypeptidase IV (DPP IV) staining reaction has demonstrated enzymatic heterogeneity of the arterial and venous segments of capillaries in rat skeletal muscle. This study compared the staining reactions of skeletal muscles in many commonly used laboratory animals, including the axolotl, chick, quail, Monodelphys, rat, mouse, hamster, guinea pig, rabbit, dog, monkey, and human. DPP IV activity was found in the venous ends of the capillaries and in the endothelium of some larger veins in many of the species but was never demonstrated in the arterial side of the circulation. AP was found in the arterial ends of capillaries in all species except the axolotl, and it was also found in the endothelium of larger arteries of most species. AP activity was absent in venous endothelium of all species except for birds and Monodelphys. DPP IV activity was found in the perineurium of intramuscular nerves of most species, and AP activity was commonly seen in tendons and intramuscular connective tissue. The interspecies variability found in this study shows that care must be taken in comparing experimental data involving this technique from one species to another, but within a species the technique allows a fine level of discrimination between functionally distinct compounds of skeletal muscle tissue.