Deficient lysosomal carboxypeptidase activity in galactosialidosis

In the lysosome, the glycosidases neuraminidase (EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23) are associated to a 52 kDa "protective protein" to form a large multi-enzymatic complex. Deficient synthesis or inactivation of this protective protein causes galactosialidosis, a lysosomal storage disorder in man in which both neuraminidase and beta-galactosidase activities are deficient. Since the protective protein possesses extensive sequence homology with carboxypeptidase Y (carb Y) and the KEX 1 gene product from yeast, we have used the artificial substrate N-CBZ-Phe-Leu to detect and characterize the peptidase activity of the lysosomal carboxypeptidase (carb L). Using both a purified preparation of the lysosomal multi-enzymatic complex and cultured skin fibroblasts of patients affected with galactosialidosis, we demonstrate that the 52 kDa protective protein is responsible for carb L activity. The fibroblasts of three patients affected with late infantile and juvenile galactosialidosis were found to be deficient in carb L activity (1.4% of normal mean value).     

Fate of HL-A antigens in aging cultured human diploid cell strains : II. Quantitative absorption studies

The expression of histocompatibility antigens on cultured human fibroblasts was studied utilizing a quantitative microabsorption assay. Trypsin treatment of cultured human embryonic and adult fibroblasts did not change their capacity to absorb selected HL-A alloantisera as compared with cells harvested by scraping. The density of HL-A antigens was found to remain unchanged throughout the finite in vitro lifetime of two human embryonic diploid cell strains (WI-38 and WI-26) and ten adult skin fibroblast cultures. Cultured fibroblasts derived from skin, lung, heart, and liver of one donor showed similar quantitative expression of HL-A1, 9, W5 and W16. These experiments support the contention that the HL-A marker system is at present the only system by which human fibroblasts derived from different normal human donors can be distinguished in vitro.     

Galactosialidosis: simultaneous deficiency of esterase, carboxy-terminal deamidase and acid carboxypeptidase activities

Esterase and deamidase activities at pH 7.0 and carboxypeptidase activity at pH 5.7 were markedly low or deficient in seven galactosialidosis fibroblast strains with deficient activity of "protective protein" for lysosomal beta-galactosidase and neuraminidase. No simultaneous deficiency of these three enzyme activities was observed in other lysosomal disease fibroblasts examined in this study. This result strongly suggests that "protective protein" is identical with a multifunctional protein with esterase/deamidase/carboxypeptidase activities and its mutation in galactosialidosis results in deficiency of these three enzyme activities.     

Human lung fibroblasts inhibit macrophage inflammatory protein-1alpha production by lipopolysaccharide-stimulated macrophages

We investigated the effect of interaction between lung fibroblasts and macrophages on macrophage inflammatory protein 1alpha (MIP-1alpha) production by macrophages. In a co-culture system consisting of WI-38 lung fibroblasts layered over THP-1 macrophages stimulated with lipopolysaccharide (LPS), MIP-1alpha production by THP-1 was significantly lower in co-culture with WI-38 than in THP-1 alone. Treatment with conditioned medium generated from WI-38 (CM-WI-38) suppressed MIP-1alpha production and mRNA expression in THP-1 cells. Such inhibitory effect of CM-WI-38 on MIP-1alpha production was abrogated by treatment with indomethacin, NS-398 (a specific COX-2 inhibitor), or anti-prostaglandin E(2) antibody. Furthermore, even in a transwell filter system separating both types of cells, co-culture-induced reduction of MIP-1alpha production was observed. Therefore, soluble factors such as prostaglandin E(2) released from lung fibroblasts are responsible for the co-culture-induced inhibition of macrophage-derived MIP-1alpha production, suggesting that immune and inflammatory cell interactions can contribute to the modulatory mechanisms involved in the regulation of the inflammatory or fibrotic process.     

Inhibition by collagen chains of lysyl hydroxylase of chick embryo and of WI-38 human lung fibroblasts.

Lysyl hydroxylase from chick embryos was strongly inhibited by heat-denatured collagens from various vertebrate sources, and by separated a chains and β components of rat tail tendon collagen. The kinetics exhibited with this enzyme when heat-denatured calf or rabbit skin collagens was used showed a mixed type of inhibition. On the other hand, a preparation of homologous heat-denatured 4,5-³H-L-lysine-labeled collagen, in itself an extremely poor substrate for the hydroxylase, showed non-competitive inhibition with a K_i of about 8–9 μM. Finally, the lysyl hydroxylase preparations from WI-38 fetal human lung fibroblasts and from transformed WI-38 cells (WI-38 VA 13) were also inhibited by heat-denatured collagens or, where tested, by separated collagen chains.     

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.     

Production of both interstitial and basement membrane procollagens by fibroblastic WI-38 cells from human embryonic lung

The synthesis and secretion of type IV procollagen, in addition to that of procollagen types I and III, was detected in cells derived from human embryonic lung (WI-38) by immunofluorescence, metabolic labeling, immunoprecipitation, collagenase digestion and the characteristic polypeptide sizes of both intact procollagen type IV chains and their initial pepsin-resistant fragments as determined by polyacrylamide gel electrophoresis. Locally obtained human embryonic lung cells secreted the same procollagens, but neither embryonic nor adult human skin fibroblasts were found to secrete type IV procollagen in amounts detectable by the same methods.     

Ultrastructural localization of plasma membrane-associated urokinase- type plasminogen activator at focal contacts

We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.     

Expression of cDNA encoding the human "protective protein" associated with lysosomal beta-galactosidase and neuraminidase: homology to yeast proteases

The "protective protein" is a glycoprotein that associates with lysosomal beta-galactosidase and neuraminidase and is deficient in the autosomal recessive disorder galactosialidosis. We have isolated the cDNA encoding human "protective protein". The clone recognizes a 2 kb mRNA in normal cells that is not evident in fibroblasts of an early infantile galactosialidosis patient. The cDNA directs the synthesis of a 452 amino acid precursor molecule that is processed in vivo to yield mature "protective protein," a heterodimer of 32 kd and 20 kd polypeptides held together by disulfide bridges. This mature form is also biologically functional since it restores beta-galactosidase and neuraminidase activities in galactosialidosis cells. The predicted amino acid sequence of the "protective protein" bears homology to yeast carboxypeptidase Y and the KEX1 gene product. This suggests a protease activity for the "protective protein."     

gamma-Glutamyltransferase in human diploid fibroblasts and other mammalian cells.

gamma-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S3 and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that gamma-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme.     

The effect of saffron on intracellular DNA, RNA and protein synthesis in malignant and non-malignant human cells.

Extract of saffron (Crocus sativis) has previously been shown to inhibit colony formation and cellular DNA and RNA synthesis by HeLa cells in vitro. In order to compare the sensitivity of malignant and non-malignant cells to saffron, we examined the effect of the extract on macromolecular synthesis in three human cell lines: A549 cells (derived from a lung tumor), WI-38 cells (normal lung fibroblasts) and VA-13 cells (WI-38 cells transformed in vitro by SV40 tumor virus). We found that the malignant cells were more sensitive than the normal cells to the inhibitory effects of saffron on both DNA and RNA synthesis. There was no effect on protein synthesis in any of the cells.     

Decreased cAMP levels in human diploid cells exposed to cholinergic stimuli.

Carbachol at a concentration of 1 micron caused very significant decreases in PGE1 stimulated cAMP levels in three human diploid lung fibroblasts, WI-38, MRC-5, and IMR-90. Detailed studies with WI-38 cells showed that carbachol acted at low concentrations and very rapidly, cAMP levels being reduced by 50% in about 1 minute. The effect was moderately reduced but still very significant in Ca++ free medium containing 10(-5)M EGTA. Acetylcholine (in the presence of physostigmine), pilocarpine, and muscarine lowered cAMP levels at concentrations of 1 or 10 micron. The effects of carbachol were exquisitely sensitive to atropine but were unaffected by hexomethonium or d-tubocurarine. These data suggested that the cholinergic response of WI-38 cells was of the muscarinic type.     

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.     

The effect of dexamethasone on the functioning of C3 receptor sites on the surfaces of human fibroblasts

The presence of C₃ receptor sites on the cell surfaces of WI-38 fibroblasts was reported in a previous paper. Here the effect of dexamethasone sodium sulfate of C₃ receptor site function was studied. The incubation of WI-38 fibroblasts with dexamethasone sodium sulfate produces the biphasic mode of action, i.e., the growth-stimulating phase with low doses (90–230 μg/ml) and the growth-inhibiting phase with high doses (450–900 μg/ml). The function of C₃ receptor sites on WI-38 fibroblasts seems to be very stable and cannot be suppressed by the pretreatment of WI-38 fibroblasts with dexamethasone in high concentrations, where the cell growth is inhibited.     

Inhibition of cellular transition from G1-resting to G1-prereplicative phase by aminonucleoside of puromycin.

Human embryonic lung fibroblasts (IMR-90 and WI-38) were arrested in the G1 phase of the cell cycle by serum deprivation and high population density. Within 1 hr after the addition of medium containing fresh serum, these cells showed an increase in rRNA synthesis. The inclusion of 100 micrograms per ml aminonucleoside of puromycin (AMS) in the fresh medium eliminated the serum stimulation of rRNA synthesis and prevented the cells from making the G1-resting phase to G1-prereplicative phase transition. AMS also prevented the synthesis of HnRNA normally found within 10 hr after serum stimulation. Serum-stimulated RNA synthesis in starved, SV-40 transformed fibroblasts (WI-38-VA-13 cells) was inhibited, but not completely prevented, by AMS indicating that transformed cells may produce specific RNA's that are not AMS-sensitive and that may be responsible for the failure of transformed cells to be arrested in G1.     

Enhancement of mucus accumulation in a human gastric scirrhous carcinoma cell line (KATO-III) by fibroblasttumor cell interaction

Human fibroblasts (WI-38 cells) were found to enhance mucus accumulation by human scirrhous carcinoma cells (KATO-III cells). Coculture of KATO-III with WI-38 cells resulted in enlargement of the KATO-III cells and increases in the proportions of PAS- and colloidal iron-positive KATO-III cells. These morphological alterations were reversed when the KATO-III cells were again cultured without WI-38 cells. Conditioned media from cultures of WI-38 cells or cocultures of KATO-III and WI-38 cells induced the same morphological alterations in KATO-III cells, suggesting that WI-38 cells produce a factor or factors that enhance mucus accumulation in KATO-III cells. This factor seemed to be a protein with a molecular weight of more than 10,000 daltons.     

γ-glutamyltransferase in human diploid fibroblasts and other mammalian cells

Summary γ-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S₃ and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that γ-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme. Preliminary reports of some of this work were presented at meetings of The American Society of Biological Chemists in Minneapolis (Abstracts Fed. Proc. 33: 957, 1974) and at Atlantic City (Abstracts Fed. Proc. 34: 2243, 1975). This work was supported by Grant NIH 1 P01 HD 07173. The WI-38 starter cultures and cell pack used in these studies were obtained through Contract M01 HD 42828 to Stanford University from the National Institute of Aging.