Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.     

Biosynthesis and intracellular transport of alpha-glucosidase and cathepsin D in normal and mutant human fibroblasts

In order to study the intracellular localization of the proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux-de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219,965-970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly-Phe-NH-Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes alpha-glucosidase and beta-hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20-30%. In contrast, treatment with Gly-Phe-NH-Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of alpha-glucosidase and cathepsin D in fibroblasts was studied by pulse-labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly-Phe-NH-Nap prior to immunoprecipitation, 70-80% of all proteolytically processed forms of metabolically labelled alpha-glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose-6-phosphate-specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, alpha-glucosidase was detected only after 2-4 h. When a pulse-chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of alpha-glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled alpha-glucosidase to the lysosomes. However, the early processing steps in which the 110-kDa precursor is converted to the 95-kDa intermediate form of the enzyme were delayed, a transient 105-kDa form was observed and the conversion of the 95-kDa intermediate form to the 76-kDa mature form of the enzyme was completely inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

Imino acid transport in human diploid fibroblasts.

These studies describe the transport of proline and hydroxyproline in human diploid fibroblasts. Inhibition and kinetic analysis demonstrate that proline is actively transported by the “A” neutral amino acid carrier. Proline transport is Na⁺ dependent and is particularly sensitive to sulfhydryl inhibitors and ouabain. Hydroxyproline is also actively transported but its transport is mediated by a system different from those described previously for other neutral amino acids. Hydroxyproline transport requires the presence of Na⁺ and is sensitive to sulfhydryl inhibitors and ouabain. There is little inhibition of hydroxyproline transport in the presence of other amino acids with the exception of methionine. The methionine inhibition of hydroxyproline transport is of the non-competitive type. Little cross-reactivity was exhibited by the systems which transport proline and hydroxyproline. These studies indicate that human skin fibroblasts do not possess an iminoglycine transport system as has been described for many other tissues. The iminoglycine transport system has been identified as the genetic transport defect in iminoglycinuria. Consequently, skin fibroblasts are not an appropriate system for use in diagnosis of this disorder.     

Potassium transport in human erythrocytes: evidence for a three compartment system

Whole human blood is incubated for periods of ½ to 3 hours with K⁴² at 37°C. At the close of this period, called pre-incubation, the plasma is removed from the cells and the cells, now become radioactive, are again incubated in a mixture of plasma and buffer for periods of up to 10 additional hours. The time course of the K⁴² activity of the incubating medium is followed. Characteristically, after 2 hours of pre-incubation, the activity in the medium rises to a peak about 1 and ½ hours after resuspension, and then falls slowly until at 10 hours it is very close to its initial value at the beginning of the resuspension interval. This transient rise in K⁴² activity in the medium is taken to indicate that the red cell does not consist of a single uniform K compartment, but contains at least two compartments. Thus one cellular compartment contains a reservoir of high specific activity K which provides the specific activity gradient necessary to drive the K⁴² content of the medium to its transient peak. Experiments with Na indicate that its behavior in this respect is unlike that of K. The experimental data are matched to a simple model system which is capable of theoretical analysis with the aid of an analogue computer. The model system, whose characteristics agree fairly well with those observed experimentally on red cell suspensions, comprises two intracellular compartments, one containing 2.35 m.eq. K/liter blood, and the other 44.1 m.eq. K/liter blood. The plasma K content is 2.64 m.eq./liter blood. The flux between plasma and the smaller intracellular compartment is 0.65 m.eq. K/liter blood hour; that between the smaller and the larger intracellular compartment, 1.77 m.eq. K/liter blood hour; and that between the larger intracellular compartment and the plasma is 0.34 m.eq. K/liter blood hour.     

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.     

L-proline uptake in human fibroblasts: evidence for a high-affinity system in addition to system A

Proline uptake was studied in human skin fibroblasts by simultaneous running of kinetic and inhibition experiments on the same cell lines. Two systems for proline uptake were shown: a high-affinity system not inhibited by alpha-(methylamino)isobutyric acid and a low affinity system inhibited by this amino acid (i.e. system A). These results appear to be of interest, firstly because up till now, system A was considered preferable for proline uptake in human fibroblasts, and secondly because they illustrate the need for combined inhibition and kinetic studies of amino acid uptake, especially when the substrate concentration range used and the respective Km of the systems do not allow their detection by kinetic analysis alone. Furthermore, this high-affinity system may have major physiological implications.     

Amino acid transport by prosthecae of Asticcacaulis biprosthecum: evidence for a broad-range transport system

Prosthecae purified from cells of Asticcaulis biprosthecum possess active transport systems that transport all 20 amino acids tested. Using ascorbate-reduced phenazine methosulphate in the presence of oxygen, all 20 amino acids are accumulated against a concentration gradient by isolated prosthecae. Results of experiments testing the inhibition of transport of one amino acid by another, and of experiments testing the exchange of exogenous amino acids with those preloaded in prosthecae, along with characteristics of mutants defective in amino acid transport, suggest the presence in prosthecae of three amino acid transport systems. One, the general or G system, transports at least 18 of the 20 amino acids tested. Another system, referred to as the proline or P system, transports seven amino acids (including proline) that are also transported by the G system. The third system transports only glutamate and aspartate, and is referred to as the acidic amino acid transport system or A system.     

Subcellular distribution of acid alpha-glucosidase in fibroblasts and of antigenically cross-reactive material in Pompe's disease fibroblasts

From fibroblasts of two cases of Pompe's disease (acid alpha-glucosidase deficiency), one of the childhood type (RH-SF-1) and one of the adult type (RH-SF-2), and normal fibroblasts, antigenically cross-reactive material and acid alpha-glucosidase were immunoprecipitated and analysed by immunoelectrotransfer blotting. The acid alpha-glucosidase and antigenically cross-reactive material (which reacts with antibody raised against normal acid alpha-glucosidase) revealed a precursor form of molecular weight 97,000 and two major components of 79,000 and 76,000. When monensin was added to the fibroblast culture, the two major components of normal acid alpha-glucosidase were decreased, whereas the large molecular weight precursor was increased. On the other hand, the 97,000 molecular weight component of cross-reactive material in the Pompe's fibroblasts (RH-SF-1 and RH-SF-2) was only slightly increased on monensin treatment. The fibroblasts were pulse-chase labelled with [2-H3] mannose and 32Pi. The cross-reactive material and acid alpha-glucosidase were precipitated with anti acid alpha-glucosidase antibody, and after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography was performed. The radiolabel of 3H in the cross-reactive material of RH-SF-1 and -2 was weak, and 32P in the cross-reactive material of both fibroblasts was very weak when compared with those of the acid alpha-glucosidase. The radiolabel of 32P in the cross-reactive material of RH-SF-1 was extremely weak. Immunofluorescence histochemistry revealed a granular localization of acid alpha-glucosidase in the normal fibroblast cytoplasm, and a diffuse distribution of cross-reactive material in the cytoplasm of RH-SF-1 and -2. Immuno-electron microscopic examinations showed a normal acid alpha-glucosidase localization on the inner side of the lysosomal membrane and also diffusely in the lysosome; when treated with monensin, it was present on the trans part of the Golgi apparatus. Antigenically cross-reactive material, however, was found in the cytoplasm and endoplasmic reticulum. Some lysosomal localization was observed sporadically. Even after monensin treatment, it was not demonstrated on the Golgi apparatus.     

Intracellular supply of phospholipids for biliary secretion: evidence for a nonvesicular transport component

Phospholipids (PL) for biliary secretion could be supplied from the endoplasmic reticulum (ER) to the plasma membrane by cytosolic transfer proteins or transport vesicles. Therefore, we studied whether biliary secretions of PL and apolipoprotein A-I (apo A-I), as markers for the ER-to-Golgi vesicular transport pathway, are tightly coupled in isolated perfused rat livers with enhanced secretion (+60%) of PL after withdrawal of the cholesterol synthesis inhibitor pravastatin (0.1% of chow, fed for 7 days). Blocking agents dissociated the secretion of apo A-I and PL. Brefeldin A as well as cycloheximide inhibited biliary secretion of apo A-I (-52%; -68%), however, not of PL. Both bilirubin ditaurate and taurodehydrocholic acid reduced biliary secretion of PL (-27%; -79%), but not of apo A-I. The data support the concept that PL destined for biliary secretion bypass the vesicular transport pathway of apo A-I through the Golgi compartment, most likely via cytosolic transfer proteins.     

The molecular heterogeneity of purified human liver lysosomal alpha-glucosidase (acid alpha-glucosidase).

An α-glucosidase active at acid pH and presumably lysosomal in origin has been purified from human liver removed at autopsy. The enzyme has both α-1,4-glucosidase and α-1,6-glucosidase activities. The K_m of maltose for the enzyme is 8.9 mm at the optimal pH of 4.0. The K_m of glycogen at the optimal pH of 4.5 is 2.5% (9.62 mm outerchain end groups). Isomaltose has a K_m of 33 mm when α-1,6-glucosidase activity is tested at pH 4.2. The enzyme exists in several active charge isomer forms which have pI values between 4.4 and 4.7. These forms do not differ in their specific activities. Electrophoresis in polyacrylamide gels under denaturing conditions indicates that the protein is composed of two subunits whose approximate molecular weights are 88,000 and 76,000. An estimated molecular weight of 110,000 was obtained by nondenaturing polyacrylamide gel electrophoresis. When the protein was chromatographed on Bio-Gel P-200 it was separated into two partially resolved active peaks which did not differ in their charge isomer constitution or in subunit molecular weights. One peak gave a strongly positive reaction for carbohydrate by the periodic acid-Schiff method and the other did not. Both had the same specific activity. The enzyme was antigenic in rabbits, and the antibodies so obtained could totally inhibit the hydrolytic action of the enzyme on glycogen but were markedly less effective in inhibiting activity toward isomaltose and especially toward maltose. Using these antibodies it was found that liver and skeletal muscle samples from patients with the “infantile” form or with the “adult” form of Type II glycogen storage disease, all of whom lack the lysosomal α-glucosidase, do not have altered, enzymatically inactive proteins which are immunologically cross-reactive with antibodies for the α-glucosidase of normal human liver.     

Intracellular transport of human immunodeficiency virus

On entering a host cell, genomic components of human immunodeficiency virus (HIV) are translocated from plasma membrane to cell nucleus where the key events of the infectious process—virus genome integration into cell chromosomes and provirus formation—take place. After provirus expression, viral components move in the opposite direction, i.e., from nucleus to plasma membrane, for virus assembly. HIV translocation is provided by transport machinery of the host cell, which is strictly controlled by viral and cell proteins. Their functional activities are closely interrelated, while their interactions promote recognition and expression of translocation signals. The aim of this review is to consider functional capabilities of one of the main regulatory matrix proteins, MA. This virus-specific protein exhibits membranotropic and nucleophilic activities and controls intracellular movements of HIV throughout its life cycle. A hypothesis on the existence of two forms of MA and their functional roles is proposed. In-depth studies of intracellular targeting of HIV virions may shed additional light on intracellular transport pathways of HIV and identify new targets for anti-HIV drugs.     

Some properties of human liver acid alpha-glucosidase.

1. Albumin activates human liver acid alpha-glucosidase (alpha-D-glucoside hydrolase, EC 3.2.1.20). From the Arrhenius plot, pH-dependence and Lineweaver-Burk plots it can be concluded that this activation is not only due to stabilisation of the enzyme, but also influences the enzymatic activity. It is proposed that for optimal functioning human liver acid alpha-glucosidase needs a protein environment. 2. Glycogen has a competitive inhibitory effect on the hydrolysis of 4-methylumbelliferyl-alpha-D-glucopyranoside, in contrast to maltose which exhibits a non-competitive type of inhibition. It is concluded that two catalytic sites exist, one for glycogen and one for maltose, while both sites influence each other. With glycogen as substrate a break in the Arrhenius plot is found. This is not the case when maltose is used as substrate. 3. The effect of antibody raised against human liver acid alpha-glucosidase on the activity of human liver acid alpha-glucosidase is studied. No corss-reacting material could be demonstrated in the liver of a patient with glycogen storage disease Type II (M. Pompe, acid alpha-glucosidase deficiency).     

Biosynthesis of two components of human acid alpha-glucosidase

Two acid alpha-glucosidase components of different molecular sizes (80 and 71 kDa) were separated from human placenta by DEAE-cellulose chromatography. Their catalytic properties were similar, and they showed almost the same molecular structure with regard to immunological properties and carboxy-terminal sequences, although the amino acid composition, the total hexose content, and the circular dichroism spectra were different. The pulse-chase labeling acid alpha-glucosidase with [3H]leucine revealed a processing pathway from a 110 kDa precursor to a 95 kDa intermediate form, then finally to 80 and 71 kDa mature forms. However, the sequence of appearance was different between the two mature enzymes. The 80 kDa component appeared first after chase for 5 h, and then the 71 kDa component followed. Their amounts became equal at 2 to 4 days. When ammonium chloride or leupeptin was added to the culture medium after chase for 5 h, the 71 kDa component failed to appear and the 80 kDa component was not converted to 71 kDa. It is concluded that probably only a part of the 80 kDa component is processed to form the 71 kDa component, although another possibility that cannot be excluded is that these two components are converted independently from the common intermediate 95 kDa protein.     

Intracellular protein degradation in serum-deprived human fibroblasts.

IMR90 human fibroblasts were labelled by incubation of cells for 48 h in medium containing 10% serum and [3H]leucine. The labelled protein was degraded at a rate of 1%/h during a subsequent incubation in medium with 10% serum. Incubation in medium without serum caused a transient enhancement of the degradation of endogenous protein, which was also found in cells labelled in medium without serum. The degradation of micro-injected haemoglobin was enhanced by serum deprivation in a non-transient manner. These results suggest that enhanced degradation in serum-free medium occurs only for a subpopulation of cell proteins and that it appears transient because the major part of the pool of susceptible endogenous proteins is being degraded during the first 20-30 h in serum-free unlabelled medium. Protein turnover in various cell compartments was measured by a double-labelling technique. Most of the enhanced degradation in serum-deprived cultures (73-83%) was due to breakdown of cytosolic proteins. The enhanced degradation of cytosolic proteins seemed to affect several proteins irrespective of their molecular mass or metabolic stability.     

Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.     

Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts

The 215-kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand-receptor interaction. Rather the antibody appears to cross-link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of "trapped receptors" is enhanced (t1/2 = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t1/2 = 30 min).