Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat

The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.     

Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase

The biosynthesis of pig small intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62) was studied by labelling of organ cultured mucosal explants with [35S]methionine. The earliest detactable form of the enzyme was an intracellular, membrane-bound polypeptide of Mr 225 000, sensitive to endo H as judged by its increased electrophoretic mobility (Mr 210 000 after treatment). The labelling of this form decreased during a chase of 120 min and instead two polypeptides of Mr 245 000 and 160 000 occurred, which both barely had their electrophoretic mobility changed by treatment with endo H. The Mr 160 000 polypeptide is of the same size as the mature lactase-phlorizin hydrolase and was the only form expressed in the microvillar membrane. Together, these data are indicative of an intracellular proteolytic cleavage during transport. The presence of leupeptin during labelling prevented the appearance of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence of leupeptin.     

Biochemical nature and topographic localization of epitopes defining four distinct CD45 antigen specificities. Conventional CD45, CD45R, 180 kDa (UCHL1) and 220/205/190 kDa

The biochemical nature and relative topographic localization of Ag determinants recognized on CD45 molecular complex by mAb defining four distinct Ag specificities (conventional CD45, CD45R, 180 kDa and 220/205/190 kDa) have been investigated. These Ag specificities display a differential biochemical, cellular, and histochemical distributions and are important in the definition of CD4-positive complementary functional T cell subsets and/or distinct stages of thymic maturation. Protease treatment of either CD45-positive cells or purified CD45 molecules revealed that both conventional CD45 and 180-kDa (UCHL1 epitope) Ag specificities are defined by epitopes present on a protease-resistant domain which is internal to the protease-sensitive epitopes defining both CD45R and 220/205/190-kDa Ag specificities. In addition, it is shown that carbohydrate moieties are contributing to the epitopes recognized by both the anti-180-kDa UCHL1 and the anti-220/205/190-kDa mAb. Neuraminidase treatment, which cleaves sialic acids either from N- or O-linked oligosaccharides, abrogated the reactivity of both mAb. However, N-glycanase treatment, which selectively cleaves N-linked sugars, did not affect the recognition of these two epitopes. Thus, these results demonstrate that the Ag determinants recognized by the UCHL1 and the anti-220/205/190-kDa mAb, which are topographically unrelated, are associated with sialic acids from O-linked-type oligosaccharides, emphasizing the contribution of carbohydrates to the Ag heterogeneity of CD45 molecular complex.     

Biosynthesis pathway of gp90MEL-14, the mouse lymph node-specific homing receptor

The mouse lymph node specific homing receptor gp90MEL-14 is a 95-kDa molecular mass ubiquitinated cell surface molecule involved in the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. The molecule is thought to consist of a core protein to which ubiquitin side chains are covalently bound. Recently we cloned the cDNA encoding the core protein; this cDNA clone encodes for a polypeptide with an estimated molecular mass of 37 kDa. We have studied the biosynthesis of gp90MEL-14 in an effort to explain the difference in molecular mass between the core protein and the 95-kDa mature molecule. Pulse labeling experiments show a rapid synthesis of a 70-kDa precursor form that contains high-mannose N-linked oligosaccharides. On processing of the high-mannose oligosaccharides into complex N-linked oligosaccharides, the precursor matures in a single step into the 95-kDa form. Experiments using deglycosylating enzymes and inhibitors of N-linked glycosylation demonstrate that the molecular mass of deglycosylated gp90MEL-14 is 45 kDa; extensive N-linked glycosylation is responsible for the difference in molecular mass with the mature 95-kDa form. The core protein molecular weight of in vitro transcribed and translated gp90MEL-14 cDNA is consistent with the estimated molecular mass of 37 kDa, calculated from the cDNA sequence of the core protein, and 8 to 10 kDa less than the protein molecular mass of gp90MEL-14 translated in vivo in the presence of tunicamycin (45 kDa). Inasmuch as we have ruled out glycosylation as accounting for this discrepancy, this is consistent with the addition of one ubiquitin moiety to the core protein during biosynthesis. Limited proteolysis confirms the similarity between in vitro transcribed gp90MEL-14 cDNA and the tunicamycin form of gp90MEL-14.     

Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I

Rat hepatic lipase is a glycoprotein bearing two N-linked oligosaccharide chains. The importance of glycosylation in the secretion of hepatic lipase was studied using freshly isolated rat hepatocytes. Various inhibitors of oligosaccharide synthesis and processing were used at concentrations that selectively interfere with protein glycosylation. Secretion of hepatic lipase activity was abolished by tunicamycin, castanospermine, and N-methyldeoxynojirimycin. No evidence was found by ELISA or Western blotting for secretion of inactive protein. Inhibition of secretion became apparent after a 30-min lag, corresponding to the time of intracellular transport of pre-existing protein. Simultaneously, intracellular hepatic lipase activity ws depleted. Secretion of hepatic lipase protein and activity was not affected by deoxymannojirimycin and swainsonine. Upon SDS-polyacrylamide gel electrophoresis, hepatic lipase secretion by deoxymannojirimycin- or swainsonine-treated cells showed an apparent Mr of 53 kDa and 55 kDa, respectively, which was distinct from hepatic lipase secreted by untreated cells (Mr = 58 kDa). We conclude that glycosylation and subsequent oligosaccharide processing play a permissive role in the secretion of hepatic lipase. As secretion is prevented by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin, but not by inhibitors of subsequent oligosaccharide trimming, the removal of glucose residues from the high-mannose oligosaccharide intermediate in the rough endoplasmic reticulum appears the determining step.     

Assembly and processing of the disulfide-linked varicella-zoster virus glycoprotein gpII(140).

Varicella-zoster virus (VZV) specifies the synthesis of at least four families of glycoproteins, which have been designated gpI, gpII, gpIII, and gpIV. In this report we describe the assembly and processing of VZV gpII, a structural protein of an apparent Mr of 140,000, which is the homolog of gB of herpes simplex virus. For these studies, we used two anti-gpII monoclonal antibodies which exhibited both complement-independent neutralization activity and inhibition of virus-induced cell-to-cell fusion. Pulse-chase labeling experiments identified a 124,000-Mr intermediate which was chased to the mature 140,000-Mr product when analyzed in nonreducing gels; in the presence of a reducing agent, the native gp140 was cleaved into two closely migrating species (gp66 and gp68). The biosynthesis of VZV gpII was further analyzed in the presence of the following inhibitors of glycoprotein processing: tunicamycin, monensin, castanospermine, swainsonine, and deoxymannojirimycin. All intermediate and mature forms were digested with endoglycosidases H and F, neuraminidase, and O-glycanase to further define high-mannose, complex, and O-linked glycans. Finally, the addition of sulfate residues was investigated. This characterization of VZV gpII revealed the following results. (i) gp128 and gp124 were early high-mannose forms, (ii) gp126 was an intermediate form with complex N-linked oligosaccharides, (iii) gp130 was a later intermediate with both N-linked and O-linked glycans, and (iv) the mature product gp140 contained a mixture of N-linked and O-linked glycans which were both sialated and sulfated. Further investigations indicated that gpII sulfation was inhibited by tunicamycin and castanospermine but not by deoxymannojirimycin or swainsonine. We also concluded that VZV gpII displayed many biological and biochemical properties similar to those of its herpes simplex virus homolog gB.     

Post-translational modification of the beta-subunit of the human fibronectin receptor

Monoclonal antibody DH12, directed against the beta-subunit of the fibronectin receptor recognizes a doublet of proteins (100 and 110 kDa) in Western blots of solubilized whole fibroblasts. Pulse-chase experiments with [35S]methionine in human skin fibroblasts suggested that the two proteins might be metabolically related as precursor (100 kDa) and product (110 kDa). Endo H digestion and [3H]fucose labeling suggested that maturation converted the high-mannose oligosaccharides (100 kDa) to the endoglycosidase H resistant complex type (110 kDa). This was supported by N-glycanase digestion and by chemical deglycosylation which showed a single polypeptide. Surface iodination of intact cells labeled only the presumed mature beta-subunit.     

Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions

To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N-linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions.     

The effect of castanospermine on the oligosaccharide structures of glycoproteins from lymphoma cell lines.

The effect of castanospermine on the processing of N-linked oligosaccharides was examined in the parent mouse lymphoma cell line and in a mutant cell line that lacks glucosidase II. When the parent cell line was grown in the presence of castanospermine at 100 micrograms/ml, glucose-containing high-mannose oligosaccharides were obtained that were not found in the absence of inhibitor. These oligosaccharides bound tightly to concanavalin A-Sepharose and were eluted in the same position as oligosaccharides from the mutant cells grown in the absence or presence of the alkaloid. The castanospermine-induced oligosaccharides were characterized by gel filtration on Bio-Gel P-4, by h.p.l.c. analysis, by enzymic digestions and by methylation analysis of [3H]mannose-labelled and [3H]galactose-labelled oligosaccharides. The major oligosaccharide released by endoglucosaminidase H in either parent or mutant cells grown in castanospermine was a Glc3Man7GlcNAc, with smaller amounts of Glc3Man8GlcNAc and Glc3Man9GlcNAc. On the other hand, in the absence of castanospermine the mutant produces mostly Glc2Man7GlcNAc. In addition to the above oligosaccharides, castanospermine stimulated the formation of an endoglucosaminidase H-resistant oligosaccharide in both cell lines. This oligosaccharide was characterized as a Glc2Man5GlcNAc2 (i.e., Glc(1,2)Glc(1,3)Man(1,2)Man(1,2)Man(1,3)[Man(1,6)]Man-GlcNAc-GlcNAc). Castanospermine was tested directly on glucosidase I and glucosidase II in lymphoma cell extracts by using [Glc-3H]Glc3Man9GlcNAc and [Glc-3H]Glc2Man9GlcNAc as substrates. Castanospermine was a potent inhibitor of both activities, but glucosidase I appeared to be more sensitive to inhibition.     

Maturation of human lactase-phlorizin hydrolase. Proteolytic cleavage of precursor occurs after passage through the Golgi complex.

Maturation of human intestinal lactase-phlorizin hydrolase (LPH) requires that a precursor (pro-LPH) be proteolytically processed to the mature microvillus membrane enzyme (m-LPH). The subcellular site of this processing is unknown. Using low-temperature experiments and brefeldin A (BFA), intracellular transport was blocked in intestinal epithelial cells. In Caco-2 cells incubated at 18 degrees C, pro-LPH was complex-glycosylated but not cleaved, while at 20 degrees C small amounts of proteolytically processed LPH were observed. These data exclude a pre-Golgi proteolytic event. BFA completely blocked proteolytic maturation of LPH and lead to an aberrant form of pro-LPH in both Caco-2 cells and intestinal explants. Therefore, proteolytic processing of LPH is a post-Golgi event, occurring either in the trans-Golgi network, transport vesicles, or after insertion of pro-LPH into the microvillus membrane.     

Intracellular processing of apolipoprotein J precursor to the mature heterodimer.

Circulating apolipoprotein J (apoJ) is a 70 kDa glycoprotein comprised of disulfide-linked alpha and beta subunits derived from a single precursor. Post-translational modifications that occur prior to apoJ secretion were assessed, with specific focus on carbohydrate type, the timing of proteolytic cleavage, and the importance of glycosylation on the cleavage and secretion processes. ApoJ was initially resolved as a single chain, intracellular precursor of 58 kDa which contained N-linked oligosaccharide but no O-linked oligosaccharide. The precursor was converted to an intracellular 70 kDa glycoprotein, which became the major intracellular form of apoJ prior to secretion. Maturation of the 58 kDa precursor involved conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurred at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of apoJ occurred rapidly, with a half-time of 30-35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of apoJ. The unglycosylated apoJ species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation.     

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.     

Intestinal aminooligopeptidase in diabetic BioBreed rat: altered posttranslational processing and trafficking

The structure of aminooligopeptidase (AOP), an intestinal brush-border digestive hydrolase, is abnormal in human diabetes and in the congenitally diabetic BioBreed Wistar (BB(d)) rat. Its assembly in the BB(d) rat was examined. After normal initial synthesis and assembly of immature AOP precursor (AOP(i)) with high-mannose N-linked chains in the endoplasmic reticulum (ER), processing of N-linked glycans in Golgi yielded a smaller than normal mature AOP precursor (AOP(m)) with persistence of some high-mannose N-linked chains. Deglycosylation analyses suggested that the mass difference could be attributed to a lower mass of N-linked with unaltered O-linked glycans in AOP(m) of the diabetic rat. Intrajejunal pulse-chase experiments revealed that the conversion of AOP(i) to AOP(m) occurred at 30 min of chase in normal rats but at 60-90 min in diabetic rats, reflecting delay in ER-to-Golgi transport or a slower processing of high-mannose chains. Once maximal transport to Golgi was achieved, the residence time in Golgi was shortened in diabetes. This altered processing of the precursor accounted for the altered structure of AOP in diabetes.     

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.     

Inhibition by cyanate of the processing of lysosomal enzymes

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.     

Effect of inhibitors of N-linked oligosaccharide processing on the cell surface expression of a melanoma integrin

The role of trimming and processing of N-linked oligosaccharides on the cell surface expression of the melanoma vitronectin receptor, a member of the integrin family of cell adhesion receptors, was examined by using specific glucosidase and mannosidase inhibitors. Inhibition of glucosidases I and II by castanospermine or N-methyldeoxynojirimycin delayed the vitronectin receptor alpha/beta chain heterodimer assembly and alpha chain cleavage and resulted in a decrease in the level of expression cell surface receptor. Conversely, the vitronectin receptor synthesized in the presence of the mannosidase I and II inhibitors, 1-deoxymannojirimycin and swainsonine, was transported normally to the cell surface with its alpha chain N-linked oligosaccharides in an endoglycosidase H-sensitive form. In the presence of swainsonine, time course studies of the cell surface replacement of control, endoglycosidase H-resistant receptor with an endoglycosidase H-sensitive form demonstrated a vitronectin receptor half-life of approximately 15-16 h. These studies provide evidence that the rates of assembly, proteolytic cleavage, and cell surface expression of the melanoma vitronectin receptor are dependent on the initial trimming of glucosyl residues from the alpha chain N-linked oligosaccharides.     

Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol.

Alkaline phosphatase is anchored to the membrane via glycosylphosphatidylinositol (GPI). Mannose residues of the GPI glycan are suggested to be derived from dolichol-P-mannose. In the present study we examined the effect of 2-fluoro-2-deoxy-D-glucose (F-Glc), an inhibitor of dolichol-P-mannose synthesis, on the biosynthesis and processing of alkaline phosphatase in JEG-3 cells. In control cells, a proform precursor (64.5 kDa) with a hydrophobic peptide domain at the COOH terminus was immediately processed into an intermediate form (63 kDa) by proteolytic removal of the COOH-terminal extension and replacement with the GPI anchor, and then to a mature form (66 kDa) by terminal glycosylation of its N-linked oligosaccharides. In contrast, when cells were treated with F-Glc (1 mM), the protein was synthesized as a proform of 61 kDa. The reduction in its molecular mass was mostly due to the inhibition in maturation of N-linked oligosaccharides by F-Glc. The 61-kDa proform identified by antibodies to the COOH-terminal peptide was detectable even at 3 h after the synthesis, and was gradually processed to doublet forms of 58-59 kDa which were finally secreted into the medium. None of these forms were labeled with [3H]ethanolamine and [3H]stearic acid, components of the GPI anchor, and expressed on the cell surface as a membrane-bound form. Taken together, these results suggest that the inhibition of the GPI synthesis causes a prolonged accumulation of the proform, which is then gradually processed into secretory forms by proteolytic removal of the COOH-terminal hydrophobic peptide.     

The Na-K-Cl cotransport protein of shark rectal gland. I. Development of monoclonal antibodies, immunoaffinity purification, and partial biochemical characterization.

The Na-K-Cl cotransporter mediates the coupled transport of Na, K, and Cl across the plasma membrane of many animal cell membranes. It is inhibited by loop diuretics such as furosemide, bumetanide, and benzmetanide. We have developed a panel of monoclonal antibodies directed against the 195-kDa shark rectal gland Na-K-Cl cotransport protein. Four representative antibodies (J3, J4, J7, and J25), each of which recognizes a discrete structural domain, were selected for detailed characterization. When a radiolabeled loop diuretic is bound to the cotransporter prior to solubilization, each antibody immunoprecipitates the same diuretic-protein complex. Of the four antibodies, J4 favors the native protein over the denatured one and does not bind well to proteolytic fragments; in contrast, J7 recognizes the cotransporter only after it has been solubilized. J3, J7, and J25 each recognize a unique ensemble of proteolytic fragments of the 195-kDa protein; analysis of the patterns of recognition has yielded a tentative assignment of the approximate location of the epitopes within the peptide. When the cotransport protein is treated with N-glycanase to remove N-linked oligosaccharides, its apparent mass decreases to approximately 135 kDa. The deglycosylated form is recognized by each of the antibodies except J25; this suggests that the J25 epitope is within the oligosaccharide component or in a peptide domain whose folding is disturbed by carbohydrate removal. An immunoaffinity matrix constructed with the J4 antibody permits single-step purification of the 195-kDa protein; other proteins copurify with the large glycoprotein, but none of these appear to be subunits of a stoichiometric complex. The amino acid sequence of four fragments of the 195-kDa cotransport protein is reported. Immunofluorescence and immunoelectron microscopy demonstrates, in agreement with physiological evidence, that the 195-kDa protein is distributed along the basolateral membrane and excluded from the apical membrane of the rectal gland secretory cell.     

Biogenesis of lysosomal enzymes in the alpha-glucosidase II-deficient modA mutant of Dictyostelium discoideum: retention of alpha-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of alpha-mannosidase or beta-glucosidase

The endoplasmic reticulum-localized enzyme alpha-glucosidase II is responsible for removing the two alpha-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum. Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, alpha-mannosidase and beta-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant alpha-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of alpha-mannosidase and beta-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N-acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.     

Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.