Recovery of function in Chinese hamster ovary cell mutants with temperature-sensitive defects in vacuolar acidification.

After 4 h at 41 degrees C, B3853 and M311, temperature-sensitive Chinese hamster ovary cell End1 and End2 mutants, respectively, are pleiotropically defective in endocytosis and trans-Golgi network-associated activities (Roff, C. F., R. Fuchs, I. Mellman, and A. R. Robbins. 1986. J. Cell Biol. 103:2283-2297). We have measured recovery of function after return to the permissive temperature. Based on return of normal transferrin-mediated Fe uptake and sensitivity to diphtheria toxin both mutants had restored endosomal function at 10 h; based on delivery of endocytosed lysosomal enzymes to lysosomes and normal sensitivity to modeccin both had functional late endocytic organelles at 10-12 h; and based on retention of newly synthesized lysosomal enzymes and sialylation of secreted glycoproteins both had functional trans-Golgi network at 6 h. At 10 h, M311 had recovered almost all of its ability to endocytose lysosomal enzymes; B3853 required 30 h to recover fully its ability to endocytose lysosomal enzymes. Slow recovery of mannose 6-phosphate-dependent uptake in B3853 reflected altered trafficking of cation-independent mannose 6-phosphate receptors. Although B3853 had normal amounts of receptor at 6-8 h, it had greatly diminished amounts of receptor at the cell surface. Altered trafficking was also suggested by the finding that B3853 rapidly degraded receptor that had been present before the shift to the nonpermissive temperature.

Chinese hamster ovary cell mutants with temperature-sensitive defects in endocytosis. I. Loss of function on shifting to the nonpermissive temperature.

We have isolated three independent Chinese hamster ovary cell mutants (B3853, I223, and M311) with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. Activities affected at 41 degrees C include uptake via the D-mannose 6-phosphate receptor, accumulation of Fe from diferric transferrin, uptake of alpha 2-macroglobulin, compartmentalization of newly synthesized acid hydrolases, resistance to ricin, and sensitivity to diphtheria and Pseudomonas toxins and modeccin. The three mutants also displayed decreased sialylation of some secreted glycoproteins at 41 degrees C, reminiscent of the nonconditional mutant DTG1-5-4 that showed both endocytic and Golgi-associated defects (Robbins, A.R., C. Oliver, J.L. Bateman, S.S. Krag, C.J. Galloway, and I. Mellman, 1984, J. Cell Biol., 99:1296-1308). Phenotypic changes were detectable within 30 min after transfer of the mutants to 41 degrees C; maximal alteration of most susceptible functions was obtained 4 h after temperature shift. At 39 degrees C, the mutants exhibited many but not all of the changes manifested at 41 degrees C; resistance to diphtheria and Pseudomonas toxins required the higher temperature. Analysis of cell hybrids showed that B3853 and DTG1-5-4 are in one complementation group ("End1"); M311 and I223 are in another ("End2"). In the End1 mutants, loss of endocytosis correlated with complete loss of ATP-dependent endosomal acidification in vitro; in the End 2 mutants partial loss of acidification was observed. At the nonpermissive temperature, residual levels of endocytic activity in B3853 and M311 were nearly identical; thus, we conclude that the differences measured in endosomal acidification in vitro reflect the different genetic loci affected, rather than the relative severity of the genetic lesions. The mutations in M311 and I223 appear to have different effects on the same protein; in I223 (but not in M311) the full spectrum of phenotypic changes could be produced at the permissive temperature by inhibition of protein synthesis.

Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.

Type C Niemann-Pick disease: a murine model of the lysosomal cholesterol lipidosis accumulates sphingosine and sphinganine in liver.

We have determined the levels of free sphingoid bases in livers of normal and cholesterol lipidotic Niemann-Pick type C mice. Hepatic sphingosine and sphinganine levels in affected mice (593 pmol/mg protein) were elevated more than 20-fold when compared to levels in age-matched normal mice (26 pmol/mg protein). Upon fractionation of mutant liver homogenates by differential centrifugation, most of the sphingoid bases sedimented with beta-hexosaminidase in the 9000 x g pellet. Co-sedimentation of sphingoid bases with a lighter beta-hexosaminidase peak in Percoll gradients suggests that these bases accumulate in lipid laden lysosomes. A cytosolic sphinganine kinase is the first enzyme in the degradative pathway of sphingoid base metabolism. Activity of this enzyme was partially deficient in crude mutant liver cytosolic extracts due to the presence of an inhibitory substance. Following molecular sieving of mutant cytosolic extracts on Sepharose 4B, sphinganine kinase, with normal levels of activity, was resolved from a complex higher-molecular-weight inhibitor fraction. The Km values for either sphinganine or ATP-Mg substrates with partially purified sphinganine kinase from normal and mutant mouse liver extracts, were similar. These findings indicate that accumulation of free sphingoid bases is not due to a direct inherent deficiency in the catalytic activity of sphinganine kinase. The possible cause and effect relationship between the accumulation of these endogenous hydrophobic amines and the lesion in intracellular cholesterol trafficking in Niemann-Pick type C disease is discussed.

The murine Niemann-Pick type C lesion affects testosterone production.

We have determined the effects of the Niemann-Pick type C (NPC) lesion, which impairs transport of cholesterol from lysosomes, on the androgenic status of male NPC mice. The mice have low serum testosterone levels resulting from decreased testosterone secretion. Testosterone secretion is reduced in NPC mouse testes incubated with 8-bromo-cAMP, 20 alpha-hydroxycholesterol, and pregnenolone compared to testosterone release by normal mouse testes under identical conditions. Ultrastructural examination of testes revealed a paucity of lipid droplets, extensive accumulation of inclusion bodies, and distorted endoplasmic reticulum in Leydig cells of adult NPC mice. The hypoandrogenemia caused systemic deficiencies in NPC mice. Seminal vesicles, a testosterone-responsive tissue, were underdeveloped in NPC male mice. The testosterone-responsive kidney beta-glucuronidase activity was also underexpressed. Seminal vesicle mass and beta-glucuronidase activity were increased by testosterone treatment of NPC mice. Many hepatic proteins, identified by microsequencing, were also deficient in NPC male mice. Levels of alpha 2-mu-globulin, glutathione S-transferase-pi, carbonic anhydrase-III, and selenium-binding protein increased in normal male mice during puberty, but did not increase in the NPC male mice. Based on the increases in protein expression during puberty, differential expression in males and females, and the reported involvement of androgens in regulating expression of some of these proteins, deficient expression of most of these proteins in male NPC mice appears to result from low testosterone levels. We conclude that a defect in testicular testosterone production in NPC male mice causes a pleiotropic deficiency in androgen-sensitive expression of proteins in various organs.

Niemann-Pick type-C disease: deficient intracellular transport of exogenously derived cholesterol.

NPC disease is an autosomal recessive neurovisceral storage disorder. A pleiotropic array of secondary enzymatic and storage abnormalities has in the past obscured a cohesive understanding of the underlying metabolic basis of this disorder. Recent findings, reviewed in this report, demonstrate that NPC disease is a cholesterol lipidosis resulting from defective intracellular cholesterol transport. The sequence of cellular events characteristic of NPC is 1) deficient intracellular transport of exogenously derived cholesterol resulting in retarded induction of cellular cholesterol homeostatic regulation; 2) accumulation of cholesterol in lysosomes; and 3) secondary cellular effects. Retarded esterification of exogenous cholesterol and accumulation of unesterified cholesterol in lysosomes is tightly coupled to the primary defect and serves as the basis for biochemical diagnosis of NPC.

Endogenous lectins from cultured cells. Isolation and characterization of carbohydrate-binding proteins from 3T3 fibroblasts.

Extracts of cultured 3T3 fibroblasts, obtained by homogenization and Triton X-100 solubilization, were fractionated on Sepharose columns covalently derivatized with asialofetuin. Three distinct carbohydrate-binding proteins (CBPs) were purified from the material bound to the affinity column: CBP35 (Mr = 35,000), CBP16 (Mr = 16,000), and CBP13.5 (Mr = 13,500). These CBPs were similar in several key properties. (a) They showed agglutination activity when assayed with rabbit erythrocytes; (b) they all appear to specifically recognize galactose-containing glycoconjugates; (c) they have low isoelectric points, pI 4.5-4.7; (d) their binding activities are rapidly lost in the absence of beta-mercaptoethanol; (e) the CBPs do not interact with each other, and the fractionated proteins can bind to asialofetuin independent of associated polypeptides; and (f) none of the proteins tend to self-associate to form oligomers of identical subunits. Comparisons of these and other properties of the CBPs suggest that CBP16 and CBP13.5 may be the murine counterparts of lactose-specific lectins previously identified in electric eel and in several bovine and avian tissues. In contrast, it appears that CBP35 represents a newly identified protein capable of binding to galactose-containing carbohydrates.

The effect of mannose 6-phosphate on the turnover of the proteoglycans in the extracellular matrix of human fibroblasts.

Human fibroblasts (SL66) were cultured in medium containing 35SO2-4 to label the glycosaminoglycans (GAGs). The cells were then detached from the culture dish to leave radioactively-labeled components of the extracellular matrix, hereafter termed 35S-labeled substrate-attached material. When unlabeled SL66 fibroblasts were plated onto this 35S-labeled substrate-attached material, the cells mediated two distinct events: (a) release of radioactivity from the substrate-attached material into the medium; (b) degradation of certain glycosaminoglycans into radioactive components of very low molecular weight including free radioactive sulfate. In the presence of mannose 6-phosphate, however, the degradation of the substrate-attached material by SL66 cells was partially inhibited. Analyses of this effect in terms of the dose-response curve, saccharide specificity, ammonium chloride sensitivity, and the requirement for cells suggest that both an intracellular compartment and the mannose 6-phosphate receptor that binds lysosomal enzymes at the cell surface may play important roles in the turnover and degradation of certain proteoglycans in substrate-attached material.

Identification of carbohydrate-binding proteins from mouse and human fibroblasts.

Three carbohydrate-binding proteins (Mr 35 000, 16 000 and 13 500) were isolated from extracts of mouse 3T3 fibroblasts by affinity chromatography on polyacrylamide beads to which was covalently bound the ligand 6-aminohexyl 4-beta-D-galactosyl-2-acetamido-2-deoxy-beta-D-glucopyranoside. None of these proteins bind to polyacrylamide beads coupled with either 6-aminohexanol or 6-aminohexyl beta-D-galactopyranoside. Therefore they appear to be carbohydrate-binding proteins specific for galactose-terminated glycoconjugates. A carbohydrate-binding protein was also purified from extracts of human foreskin fibroblasts. This protein (Mr 35000) may represent the human counterpart of the mouse protein of similar Mr and binding properties.

The effect of mannose6-phosphate on the turnover of cell surface glycosaminoglycans.

Human fibroblasts (SL66) were cultured in medium containing 35SO4(2-) to label the glycosaminoglycans (GAGs). After washing, the labeled cells were chased in the presence or absence of mannose6-phosphate (M6P) and the GAGs were analyzed in terms of three arbitrary fractions: 1, Extracellular (soluble medium), 35S radioactivity higher in cultures without M6P than in cultures with M6P. 2, Pericellular (cell surface-associated), 35S radioactivity lower in cultures without M6P than in cultures with M6P. 3, Intracellular (residue within the intact cell), no difference in 35S radioactivity between the two sets of cultures. In addition, when the 35S-labeled GAGs from corresponding cellular compartments derived from cultures with and without M6P were digested with pronase and chondroitin ABC lyase, and then compared by chromatography on Sepharose CL-6B, distinct molecular differences in both the extracellular and pericellular fractions were observed. Several lines of evidence indicate that the effect of M6P on the turnover of 35S-labeled GAGs in our assay system reflects disruption of cell surface lysosomal enzyme activity. For example, when the experiment was performed with I cells, which lack enzymes carrying the M6P marker, no difference was seen in cultures with or without M6P. The addition of lysosomal enzymes derived from normal human fibroblasts to 35SO4-labeled I cells, however, resulted in the turnover of pericellular GAGs and this effect was inhibited by M6P. These results suggest that one possible function of cell surface receptors recognizing the M6P moiety of lysosomal enzymes is to anchor certain of these enzymes proximate to their substrates at the cell surface.

Deficiencies in sex-regulated expression and levels of two hepatic sterol carrier proteins in a murine model of Niemann-Pick type C disease.

Hepatic sterol carrier protein-2 (SCP2) and sterol carrier protein-X (SCPx) levels in normal and in mutant Niemann-Pick Type C mice were determined by immunoblotting with antiserum against rat SCP2. A 14-kDa protein (SCP2) was detected in the cytosol fraction and a 58-kDa protein (SCPx) was found in both cytosolic and organellar fractions. Expression of hepatic SCPx protein was developmentally regulated in a sex-specific pattern. The amounts of organelle-associated SCPx increased 4-fold during sexual development of normal males but decreased dramatically during development of normal females. Levels of hepatic SCP2 increased much less dramatically during sexual maturation of normal males and females. Adult Niemann-Pick Type C mice were deficient in both hepatic SCPx and SCP2. The deficit in SCPx in affected males reflected a failure to increase hepatic SCPx levels during sexual maturation. In affected males SCPx remained at levels found in immature mice. Affected male and female mice were also unable to maintain levels of hepatic SCP2. The level of SCP2 was near normal in affected immature males and subnormal in affected immature females. During sexual maturation hepatic SCP2 declined in affected animals.

Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport.

Niemann-Pick Type C (NPC) disease is a cholesterol lipidosis resulting from defective postlysosomal cholesterol transport. In normal cells this segment of cholesterol trafficking is inhibited by treatment with either U18666A or imipramine. Other compounds are also capable of blocking postlysosomal cholesterol transport: stearylamine, RV-538, and sphinganine inhibit low-density lipoprotein-induced esterification of cholesterol and cause unesterified cholesterol to accumulate in perinuclear vesicles. These vesicles can be stained with filipin to give a staining pattern indistinguishable from that seen in NPC fibroblasts. Because all of these compounds are hydrophobic amines, we conclude that most, if not all, hydrophobic amines block the postlysosomal transport of cholesterol. These results also raise the possibility that an endogenous amine, e.g., sphinganine, may inhibit cholesterol transport in NPC.

Progesterone blocks cholesterol translocation from lysosomes.

Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.

Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Quantification of active caspase 3 in apoptotic cells.

We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying relative amounts of active caspase 3 in apoptotic cells. Covalent modification of caspase 3 active sites with a biotinylated inhibitor differentiates active from latent caspases. Capture on an ELISA plate with an antibody specific for caspase 3 makes the assay specific for caspase 3. Detection is with horseradish peroxidase (HRP)-conjugated streptavidin that binds to the biotinylated inhibitor covalently bound to caspase 3. Using the assay we detected 6.6 ng active caspase 3 per 10(6) apoptotic staurosporine-treated Jurkat cells. Specificity of the assay for caspase 3 was demonstrated by lack of signal with purified caspases 2, 7, 8, and 10 that were modified by a biotinylated inhibitor. Specificity was also demonstrated by lack of signal with apoptotic MCF-7 cells which do not express caspase 3. The ability to discriminate between active and latent caspase 3 was shown by Western blotting with HRP-streptavidin and anti-caspase 3. Although latent caspase 3 was captured it was not covalently modified with the biotinylated inhibitor. The basic principle of using a covalent inhibitor to identify active enzymes and an antibody to differentiate between enzymes with similar activities has potential for quantifying active members of many classes of enzymes.

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.