Intercellular exchange of lysosomal hydrolases between mutant human fibroblasts and other cell types

Human fibroblasts with a genetic deficiency of a single lysosomal enzyme and fibroblasts from a patient with ‘I-cell’ disease with a multiple deficiency of lysosomal hydrolases were used as recipient cells in studies on recognition and uptake of β-N-acetylhexosaminidase (hexosaminidase), β-glucuronidase and β-galactosidase. Normal human fibroblasts, and fibroblasts, hepatocytes and hepatoma cells from the rat were used as donor cells. The release of hexosaminidase was found to be similar among these different cell types, but the extracellular activities of β-glucuronidase and β-galactosidase were much higher in the rat cell cultures than in cultures of normal human fibroblasts. The enzymes released by rat fibroblasts were ingested by deficient human fibroblasts; enzyme from normal human fibroblasts was shown to be taken up by rat fibroblasts by means of electrophoresis. This indicates that reciprocal transfer of lysosomal hydrolases occurs between human and rat fibroblasts. Rat hepatocytes released hydrolases that were poorly taken up by human recipient fibroblasts and uptake of human fibroblast enzyme was not detected in the hepatocytes. Rat hepatoma cells, on the other hand, released lysosomal enzymes that were taken up by human deficient cells with a higher efficiency than those from fibroblasts. The uptake was subject to competitive inhibition by mannose 6-phosphate, the kinetics of which were comparable with those reported for ‘high-uptake’ forms of lysosomal enzymes [1–2]. Electrophoretic studies showed that rat hepatoma cells were not only capable of ingesting hexosaminidase from normal human fibroblasts, but also defectively processed enzyme [4–5] released by ‘I-cells’. These findings make rat hepatoma cells a useful model for the study of recognition and uptake of lysosomal enzymes.     

Identification of rabbit and mouse β-glucuronidases in human fibroblasts following direct interaction with lymphocytes

Human fibroblasts totally deficient in β-glucuronidase acquired high levels of enzyme activity when co-cultured with mouse or rabbit lymphocytes. Direct cell-to-cell contact was obligatory for this process. The enzyme acquired by the fibroblasts was shown to be identical to β-glucuronidase from donor lymphocytes by its position of elution from DEAE-cellulose, thermal stability, mobility on polyacrylamide gels and by its antigenic determinants. The enzyme extracted from deficient fibroblasts after co-culture with lymphocytes showed no evidence of any hybridisation between human and mouse or rabbit sub-units. It is concluded that during direct cell interaction, enzymically active β-glucuronidase is transferred directly from donor lymphocytes to deficient fibroblasts by a mechanism, previously shown not to involve normal receptor mediated endocytosis.     

Long - lasting synchrony of the division of enteric bacteria

Recent finding of α-N-acetylglucosamine(1)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of mannose 6-phosphate residues involves transfer of N-acetylglucosamine 1-phosphate to mannose. Using dephosphorylated β-hexosaminidase as acceptor and [β-³²P]UDP-N-acetylglucosamine as donor for the phosphate group, phosphorylation of β-hexosaminidase by microsomes from rat liver, human placenta and human skin fibroblasts was achieved. The reaction was not affected by tunicamycin. Acid hydrolysis released mannose 6-[³²P]phosphate from the phosphorylated β-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine. The transferase activity was deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in l-cell disease.     

DNA primase of plasmid ColIb is involved in conjugal DnA synthesis in donor and recipient bacteria.

The sog gene of the IncI alpha group plasmid ColIb is known to encode a DNA primase that can substitute for defective host primase in dnaG mutants of Escherichia coli during discontinuous DNA replication. The biological significance of this enzyme was investigated by using sog mutants, constructed from a derivative of ColIb by in vivo recombination of previously defined mutations in a cloned sog gene. The resultant Sog- plasmids failed to specify detectable primase activity and were unable to suppress a dnaG lesion. These mutants were maintained stably in E. coli, implying that the enzyme is not involved in vegetative replication of ColIb. However, the Sog- plasmids were partially transfer deficient in E. coli and Salmonella typhimurium matings, consistent with the hypothesis that the normal physiological role of this enzyme is in conjugation. This was confirmed by measurements of conjugal DNA synthesis. Studies of recipient cells have indicated that plasmid primase is required to initiate efficient synthesis of DNA complementary to the transferred strand, with the protein being supplied by the donor parent and probably transmitted between the mating cells. Primase specified by the dnaG gene of the recipient can substitute partially for the mutant enzyme, thus providing an explanation for the partial transfer proficiency of the mutant plasmids. Conjugal DNA synthesis in dnaB donor cells was deficient in the absence of plasmid primase, implying that the enzyme also initiates synthesis of DNA to replace the transferred material.     

Multiple transfer of lysosomal enzymes from normal lymphocytes to I-cell disease fibroblasts

Cells from patients with inherited lysosomal deficiency diseases can acquire the missing lysosomal enzyme by direct cell-to-cell transfer from normal lymphocytes. Cells from I-Cell Disease (Mucolipidosis type II; ICD) patients are simultaneously deficient in many lysosomal enzymes due to an inborn error of glycoprotein processing. In this study we show that such cells acquire high levels of several of the missing lysosomal enzymes when they are cultured in contact with lymphocytes. Moreover, the present results also show that enzyme levels in the donor lymphocytes are not depleted but increase during cell contact with the fibroblasts.     

The distribution of hydrolytic enzyme activities in human fibroblast cultures and their intercellular transfer.

Both N-acetyl-β-D-glucosaminidase A and B (EC 3.2.1.30) are continuously secreted by normal cultured fibroblasts and can be taken up by deficient Sandhoff cells without cellular contact. The absence of intercellular transfer of β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) in cocultivations of normal and deficient fibroblasts is accompanied by very low extracellular activities of these enzymes in cultures of normal fibroblasts. For each of the hydrolases tested an appreciable amount of activity was found in the “pericellular” fraction. N-acetyl-β-D-glucosaminidase which has been taken up by deficient Sandhoff cells has an intracellular half-life of 6 days. The ingested intracellular enzyme, which is presumably localized in the lysosomes, is partly transferred to the pericellular fraction, and to the extracellular fraction. The results are discussed in relation to the secretion-recapture model proposed by Hickman and Neufeld.     

Lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact

We have examined the changes in the activities of the different types of alpha-D-mannosidase when fibroblasts from patients deficient in the lysosomal form of the enzyme are cultured together with normal lymphocytes. Our results show that whereas the mannosidosis cells acquired high levels of this enzyme, the activities of both the Golgi and the endoplasmic reticulum forms of alpha-D-mannosidase remained the same as in the fibroblasts cultured alone in the absence of lymphocytes. The increase in the activity of the lysosomal enzyme in the cocultured fibroblasts was not affected by the presence of mannose 6-phosphate or alpha-methyl mannoside, inhibitors of receptor- and lectin-mediated uptake of lysosomal enzymes, respectively, but it did require cell-to-cell contact. Ion-exchange HPLC and electrophoresis in polyacrylamide gradient gels showed that the acquired enzyme had the same elution profile and molecular size as the lysosomal form of the enzyme present in the lymphocytes. Immunoprecipitation studies using antibody specific for the lymphocyte type of lysosomal alpha-D-mannosidase confirmed that the increased activity in the cocultured mannosidosis cells resulted from the acquisition of the lymphocyte enzyme. Cytochemical examination revealed, however, that the transferred lymphocyte enzyme was localized in cytoplasmic organelles in the peripheral regions of the recipient fibroblasts. These results show that lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact with mannosidosis cells.     

Correction of the biochemical defect in porphobilinogen deaminase deficient cells by non-viral gene delivery

Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88–200 fold in human AIP fibroblasts and synthesis of PP was increased from 21–152% of normal after ALA incubation. Similar results were obtained in mouse PBGD deficient cells, although the PP levels were several-fold lower as compared to human cells. HPLC analysis confirmed that PP was the main porphyrin intermediate that was formed. Addition of porphobilinogen (PBG) resulted in 3–7 fold lower synthesis of PP as compared to ALA addition. These results show that non-viral gene delivery of plasmids encoding PBGD results in a high expression of functional PBGD shown by induced synthesis of PP in PBGD deficient cells after supplementation of ALA and PBG.     

Intercellular exchange of lysosomal enzymes: enzyme assays in single human fibroblasts after co-cultivation.

Intercellular exchange of N-acetyl-β-D-glucosaminidase (EC 3.2.1.30) β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) was studied after cocultivation of normal and enzyme deficient human fibroblasts in confluent cultures. Enzyme activities were measured in single cells using microchemical procedures. After co-cultivation of normal control fibroblasts and those from a patient with Sandhoff's disease an increase of activity of N-acetyl-β-D-glucosaminidase was found in Sandhoff cells, together with a decrease of activity in normal control cells. After co-cultivation of normal fibroblasts and those from patients with glycogenosis II and GM1-gangliosidosis, no indication was found for intercellular transfer of acid α-glucosidase and β-galactosidase respectively. The significance of the results is discussed in respect of the hypothesis of Hickman and Neufeld about secretion and uptake of lysosomal enzymes.     

Catalase in cultured skin fibroblasts from patients with the cerebro-hepato-renal (Zellweger) syndrome: normal maturation in peroxisome-deficient cells

We have compared the properties of catalase in cultured skin fibroblasts from patients with the cerebro-hepato-renal (Zellweger) syndrome, in which peroxisomes are deficient, with those of catalase in fibroblasts from control subjects. The enzymes from the two types of fibroblasts are indistinguishable with respect to kinetic properties, subunit size and molecular mass of the native enzyme. The turnover of the enzyme, measured by following the rate of reappearance of catalase activity in fibroblasts after irreversible inactivation of existing molecules by 3-aminotriazole treatment of the cells, was the same in Zellweger fibroblasts as in control cells. These findings indicate that normal maturation of catalase can occur in the soluble cytoplasm and provide an explanation for the occurrence of extra-peroxisomal catalase in tissues and cells.     

Complementation mapping in microcell hybrids: localization of Fpgs and Ak-1 on Mus musculus chromosome 2

The gene encoding folylpolyglutamyl synthetase (FPGS) was assigned to mouse chromosome 2 by complementation mapping. Chinese hamster ovary cells (AuxBl) deficient in FPGS, and consequently auxotrophic for glycine, adenosine, and thymidine (gat-), were employed as recipients in microcell-mediated chromosome transfer experiments. Mouse chromosomes derived from diploid embryo fibroblasts were introduced into hamster AuxBl cells, and gat+ microcell hybrids were selected in medium lacking adenosine and thymidine. Mouse chromosome 2 was the only donor chromosome whose presence correlated with expression of FPGS activity. Furthermore, every gat+ hybrid clone expressed murine AK-1, a marker previously assigned to chromosome 2. Eight of 20 clones analyzed retained deletion chromosomes derived from mouse chromosome 2. These clones were used to localize murine Fpgs and Ak-1 to a region of this chromosome, namely 2 (cen leads to Cl).     

Communication between normal and enzyme deficient cells in tissue culture

Correction of certain mutant phenotypes by intimate contact with normal cells, i.e. ‘metabolic cooperation’, is an easily studied form of cell communication. Metabolic cooperation between normal cells and mutant cells deficient in hypoxanthine-guanine or adenine phosphoribosyl transferase (HGPRTase and APRTase respectively) appears to be the result of transfer of the enzyme product, nucleotide or nucleotide derivative, from normal to mutant cells. This process shows selectivity in that mutant derivatives of mouse L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human fibroblasts with these enzyme deficiencies, exhibit correction of the mutant phenotype, when in contact with normal donor cells. There is also selectivity with respect to substances transferred, since other mutant phenotypes, i.e. G-6 PD deficiency, are not corrected by contact with normal cells. Species specificities do not appear to influence metabolic cooperation, therefore heterospecific cell mixtures provide an opportunity to cytologically distinguish cells and study individual cell interactions.     

The analysis of telomere length and telomerase activity in cloned pigs and cows

Inefficiency in the production of cloned animals is most likely due to epigenetic reprogramming errors after somatic cell nuclear transfer (SCNT). In order to investigate whether nuclear reprogramming restores cellular age of donor cells after SCNT, we measured telomere length and telomerase activity in cloned pigs and cattle. In normal pigs and cattle, the mean telomere length was decreased with biological aging. In cloned or transgenic cloned piglets, the mean telomere length was elongated compared to nuclear donor fetal fibroblasts and age-matched normal piglets. In cloned cattle, no increases in mean telomere length were observed compared to nuclear donor adult fibroblasts. In terms of telomerase activity, significant activity was observed in nuclear donor cells and normal tissues from adult or new-born pigs and cattle, with relatively higher activity in the porcine tissues compared to the bovine tissues. Cloned calves and piglets showed the same level of telomerase activity as their respective donor cells. In addition, no difference in telomerase activity was observed between normal and transgenic cloned piglets. However, increased telomerase activity was observed in porcine SCNT blastocysts compared to nuclear donor cells and in vitro fertilization (IVF)-derived blastocysts, suggesting that the elongation of telomere lengths observed in cloned piglets could be due to the presence of higher telomerase activity in SCNT blastocysts. In conclusion, gathering from the comparative studies with cattle, we were able to demonstrate that telomere length in cloned piglets was rebuilt or elongated with the use of cultured donor fetal fibroblasts.     

Cloning of functional alpha propionyl CoA carboxylase and correction of enzyme deficiency in pccA fibroblasts.

Propionyl CoA carboxylase (PPC) is a heteromeric enzyme composed of alpha subunits (PCCA) and beta (PCCB) subunits. We describe cDNA clones expressing human PCCA and complementation of the genetic defect in pccA fibroblasts by DNA-mediated gene transfer. Two cDNA clones were constructed. The first corresponds to the previously reported, putatively full-length, open reading frame. The second encodes a chimera composed of the mitochondrial leader sequence of human methylmalonyl CoA mutase and the mature PCCA protein. Both clones reconstitute propionate flux to normal levels in fibroblasts from patients genetically deficient in PCCA (pccA). The maximal level of propionate flux approached, but never exceeded, the levels seen in control plates of normal cells. In contrast, the maximal level of PPC holoenzyme activity reached only 10%-20% that of normal controls, which corresponded roughly to the fraction of cells actually transformed with the recombinant gene. These data suggest that the level of PCCA expression in fibroblasts does not normally limit PCC holoenzyme activity or propionate flux. The fact that a small fraction of cells reconstitutes propionate flux to normal levels suggests that metabolic cooperation between cells is capable of increasing the metabolic capacity of recombinant enzyme in a subpopulation of cells. These factors may have important implications for the rational design of somatic gene therapy for PCCA deficiency.     

Characterization of the biochemical basis of a complete deficiency of the adenine phosphoribosyl transferase (APRT)

Summary In order to study the biochemical basis of a complete deficiency of adenine phosphoribosyl transferase (APRT) the enzyme was purified to homogeneity, its properties were characterized, and antibodies raised. The enzyme is indirectly involved in adenine uptake. Apparently, by forming AMP the internal concentration of adenine is kept low allowing its diffusion.The same APRT is present in various tissues as was revealed by antibody inactivations employing anti-erythrocyte APRT as well as by direct enzyme assays in cells from the APRT deficient patient. In vitro cultured fibroblasts derived from this patient had less than 0.02% enzyme activity. No cross-reacting material was found in erythrocytes obtained from an APRT deficient child.     

Characterization of the mutant N-acetylglucosaminylphosphotransferase in I-cell disease and pseudo-Hurler polydystrophy: complementation analysis and kinetic studies

Complementation was examined among various types of I-cell disease and pseudo-Hurler polydystrophy by monitoring N-acetylglucosaminylphosphotransferase activity in multinucleated cells produced by fusing pair combinations of cultured skin fibroblasts. Patients with the classical forms of these disorders (5 I-cell disease and 3 pseudo-Hurler polydystrophy cell lines) comprised one complementation group and 5 cell lines from patients with variant forms of pseudo-Hurler polydystrophy comprised a distinct complementation group. In the first group, total or partial deficiency of the transferase activity was demonstrated with both natural (lysosomal enzymes) and artificial (alpha-methylmannoside) acceptor substrates with low Vmax but apparently normal Km values for the donor (UDP-GlcNAc) and acceptor (alpha-methylmannoside) substrates. The activity toward artificial substrate could be inhibited by adding exogenous lysosomal enzyme preparations to the reaction mixture. In the second group, the cells demonstrated deficiency of the transferase activity toward lysosomal enzyme acceptors but had normal activity toward alpha-methylmannoside acceptor and this activity could not be inhibited by the addition of exogenous lysosomal enzyme preparations. These findings suggest that N-acetylglucosaminylphosphotransferase is composed of at least two distinct subunits, a catalytic subunit which is absent or defective in the first complementation group, and a recognition subunit which is altered or deficient in the second group.     

Metabolic cooperation in cell culture: studies of the mechanisms of cell interaction

Metabolic cooperation is a form of cell communication in which the mutant phenotype of enzyme deficient cells, as determined by incorporation of labeled substrates, is corrected in culture by contact with normal cells. Previous studies showed that metabolic cooperation between normal and hypoxanthine phosphoribosyl transferase deficient cells (HPRT^?) was the result of transfer of product of the enzyme, nucleotide or nucleotide derivative, from normal to mutant cells rather than transfer of enzyme or informational macromolecules leading to the synthesis of the enzyme. In the present study the nature and mechanisms involved in these cell interactions were investigated. Effective communication is observed within one hour of cell contact. Modifications of the extracellular environment including changes in osmolarity, concentration of sodium and divalent ions failed to interfere significantly with transfer. Changes of cell shape induced by cyclic nucleotides, hormones and urea also did not affect communication. Cytochalasin B which dissociates microfilaments and binds to cell membranes reduced metabolic cooperation while colcemide which dissociates microtubules had little effect. Enzymatic oxidation and iodination of cell surface structures abolished metabolic cooperation. The subcellular localization of label in donor cells is important in determining efficiency of transfer. Metabolic cooperation is efficient when radioactive label is primarily located in the nucleus and inefficient if the label is cytoplasmic. Cell lines previously classified as “non-communicating” because they lack gap junctions, ionic coupling and metabolic cooperation were shown in the present study to communicate when incubated with labeled substrates for 20 hours rather than 3. Cell communication is a more generalized phenomenon among cells in contact than previously appreciated.     

Lysosomal integral membrane protein 2 (LIMP-2) restricts the invasion of Trypanosoma cruzi extracellular amastigotes through the activity of the lysosomal enzyme β-glucocerebrosidase

Lysosomal integral membrane protein 2 (LIMP-2, SCARB2) is directly linked to β-glucocerebrosidase enzyme (βGC) and mediates the transport of this enzyme from the Golgi complex to lysosomes. Active βGC cleaves the β-glycosidic linkages of glucosylceramide, an intermediate in the metabolism of sphingoglycolipids, generating ceramide. In this study we used mouse embryonic fibroblasts (MEFs) deficient for LIMP-2 and observed that these cells were more susceptible to infection by extracellular amastigotes of the protozoan parasite Trypanosoma cruzi when compared to wild-type (WT) fibroblasts. The absence of LIMP-2 decreases the activity of βGC measured in fibroblast extracts. Replacement of βGC enzyme in LIMP-2 deficient fibroblasts restores the infectivity indices to those of WT cells in T. cruzi invasion assays. Considering the participation of βGC in the production of host cell ceramide, we propose that T. cruzi extracellular amastigotes are more invasive to cells deficient in this membrane component. These results contribute to our understanding of the role of host cell lysosomal components in T. cruzi invasion.     

Dogs cloned from fetal fibroblasts by nuclear transfer

Fetal fibroblasts have been considered as the prime candidate donor cells for the canine reproductive cloning by somatic cell nuclear transfer (SCNT) in regard to the future production of transgenic dogs, mainly due to their higher developmental competence and handling advantage in gene targeting. In this study, the cloning efficiency with canine fetal fibroblasts as donor cells was determined. A total of 50 presumptive cloned embryos were reconstructed, activated and transferred into the oviducts of naturally synchronous recipient bitches. While the fusion rate (76.9%) was similar to those of our earlier studies with adult fibroblasts as donor cells (73.9–77.1%), a high cloning efficiency (4.0%; 2 births/50 embryos transferred) was found compared to the previous success rate with adult fibroblasts (0.2–1.8%). The cloned beagles were healthy and genotypically identical to the donor fibroblast cells. This study shows that a fetal fibroblast cell would be an excellent donor for future production of transgenic dogs via gene targeting in this cell followed cloning using SCNT technology.     

The effects of low-density lipoprotein and cholesterol on acyl-coenzyme A: cholesterol acyltransferase activity in membranes from cultured human fibroblasts.

Membranes prepared from cultured fibroblasts were assayed for acyl-coenzyme A: cholesterol acyltransferase (ACAT) by a method that relied exclusively on the cholesterol already present on the membranes as the sterol substrate. Changes in membrane ACAT activity during incubation of fibroblasts under a variety of conditions were similar to the changes in the rate of incorporation of oleic acid into cholesteryl esters by the intact cells. The addition of low-density lipoprotein (LDL) to fibroblasts pre-incubated with lipoprotein-deficient serum led to a transient increase in membrane ACAT activity, which reached its peak after 7h and was related to the receptor-mediated uptake and degradation of the lipoprotein by the cells. However, after incubation of the membranes with a cholesterol-rich donor lipoprotein, which resulted in an equilibration of cholesterol between membranes and donor, each preparation exhibited the same activity. In contrast with these effects of LDL, incubation of the cells with non-esterified cholesterol produced a prolonged increase in ACAT activity and an increase in the activity observed after equilibration. Furthermore, ACAT activity in cells grown with linoleic acid was higher, both before and after the addition of LDL, than that of cells grown in normal medium or with palmitate. The increase in activity produced by LDL was also greater, reflecting the greater rate of degradation of LDL by the cells, and was associated with an increase in the activity observed after equilibration with donor. The results suggest that although fibroblasts can increase the amount of active enzyme on their membranes to accommodate an exceptionally high or prolonged supply of cholesterol, under normal circumstances the increase in membrane ACAT activity produced by LDL can be explained entirely by an increase in the amount of cholesterol in the substrate pool.