Allen L. Horwitz

Regulation of Glycosaminoglycan Synthesis by Thyroid Hormone in Vitro

Human skin fibroblasts synthesize and accumulate glycosaminoglycans (GAG). Recently, we reported that fibroblasts incubated in thyroid hormone-deficient media accumulate more GAG than do cultures incubated in the same media enriched with 0.1 muM triiodothyronine (T(3)) (1981. Endocrinology. 108: 2397). The current study characterizes that enhanced accumulation. Confluent cultures were maintained in thyroid hormone-deficient media without or with added T(3), labeled with [(3)H]acetate and analyzed for total [(3)H]GAG and [(3)H]hyaluronic acid content. Addition of T(3) to thyroid hormone-depleted media consistently inhibited the incorporation of [(3)H]acetate into GAG by 28-60% in fibroblast cultures from four different normal human donors. Maximal inhibitory effect was observed within 3 d after hormone addition at concentrations > 1 nM. 73% of the maximal inhibitory effect was observed in the presence of physiologic concentrations of T(3) (0.16 nM total T(3) or 1.4 pM free T(3)). The following observations indicated that T(3) inhibition of [(3)H]GAG accumulation is most likely due to a decrease in GAG synthesis rather than to changes in the acetate pool or GAG degradation: (a) Addition of 0, 100, 500, and 2,500 muM unlabeled acetate progressively decreased [(3)H]acetate incorporation into GAG, up to 80%, without altering the further inhibitory effect of T(3) (35-40%); (b). A similar effect of T(3) on GAG (32% inhibition) was observed using [(3)H]glucosamine as substrate; (c) T(3) decreased hyaluronate synthetase activity by 32%; and (d) There was no effect of T(3) on GAG degradation in a pulse-chase experiment. The effect of T(3) on [(3)H]GAG accumulation appears to be quite specific, since the hormone had no effect on the incorporation of [(3)H]leucine into trichloroacetic acid-precipitable material.Thus, thyroid hormone inhibits GAG accumulation in a dose-, time-dependent, and reversible manner. This inhibition is apparently due to specific effects on the rate of macromolecular synthesis.

Synthesis of chondromucoprotein by chondrocytes in suspension culture

Abstract Embryonic chick chondrocytes growing in suspension culture excrete into the medium chondromucoprotein which appears to be identical to chondromucoprotein isolated from epiphyseal cartilage. Low oxygen tension favored maximum production of chondromucoprotein. The presence of ascorbic acid enhanced both chondromucoprotein and collagen synthesis. Glycosyltransferases involved in chondromucoprotein synthesis were detected in homogenates of such cultured chondrocytes.

Generalized N-Acetylneuraminic Acid Storage Disease: Quantitation and Identification of the Monosaccharide Accumulating in Brain and Other Tissues

Abstract: Brain and other tissues from a patient with extensive neonatal as‐cites and clinical symptoms suggestive of a severe neurovisceral storage disorder were examined following autopsy for the accumulation of oligosaccharides. This carbohydrate analysis revealed the presence of large amounts (3–21 μmol/g fresh weight) of sialic acid in brain, liver, and kidney tissue as the major abnormality. Exhaustive characterization of the accumulating material by gel filtration, gas‐liquid chromatography, thin‐layer chromatography, and GLC‐mass spectrometry positively identified the saccharide as free N‐acetylneuraminic acid. Based on the accumulation of only free N‐acetylneuraminic acid in the tissue of this patient, and normal activities of lysosomal enzymes involved in the catabolism of cellular glycoproteins, this storage disorder appears to result from a previously unreported defect in glycoconjugate metabolism.

The enzymic defect in Morquio's disease: the specificity of N-acetylhexosamine sulfatases.

Abstract Extracts of Morquio fibroblasts lack N-acetylgalactosamine 6-sulfate sulfatase activity, but exhibit normal levels of N-acetylglucosamine 6-sulfate sulfatase activity. Thus, the enzyme defective in Morquios disease is a sulfatase specific for the 6-sulfate linked to sugars with the galactose configuration. Hydrolysis of ester sulfate by this enzyme is limited to 6-sulfate groups occurring at the non-reducing terminal.

Molecular basis of an adult form of β-hexosaminidase B deficiency with motor neuron disease

A patient (KL) with progressive motor neuron disease associated with partial Hex A (alpha beta) and no Hex B (beta beta) activity, synthesized beta-chains which only associated with alpha-chains. To identify the molecular basis of this inability of beta-chains to self associate, RNA from cultured fibroblasts was reverse transcribed, the cDNA encoding the beta-chain amplified by polymerase chain reaction, subcloned, and sequenced to reveal two types of single missense mutation. The first mutation, (Type I) 619A----G, was paternally inherited and converted a 207IIe----Val in a highly conserved region believed to be associated with catalytic activity and activator protein binding. Biochemical evidence for impaired activator protein binding was obtained by purifying Hex A from KL urine and demonstrating a greater than 50% reduction of in vitro GM2 hydrolysis compared to normal urinary Hex A. In other cDNA species (Type II), a maternally inherited 1367A----C mutation converted 456Tyr----Ser in another highly conserved region of the beta-chain and we propose that this mutation leads to the inability of the beta-chains to self associate and thus reach maturity. These same cDNA species contained a second 362A----G mutation which converted 121Lys----Arg, but is apparently a polymorphism since it also occurs in some normal subjects. We propose that the patient is a compound heterozygote in which a combination of no self-association of the mutant beta-chains and impaired activator protein binding to alpha-beta (mutant) (Hex A) required for GM2 hydrolysis result in total beta-Hex B deficiency and slow accumulation of GM2 ganglioside, primarily in motor neurons.

N-Acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 mumol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [3H]glucosamine-HCl or N-[3H]acetylmannosamine showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.

Effect of normolipemic and hyperlipemic serum on biosynthetic response to cyclic stretching of aortic smooth muscle cells.

Arterial smooth muscle cells synthesize matrix macromolecules in response to mechanical stimulation. Exposure to serum lipids also stimulates connective tissue fiber accumulation. To assess the effect of serum lipids on the biosynthetic response to tensile stress, we subjected rabbit aortic smooth muscle cells that were cultured on purified elastin membranes to cyclic stretching and relaxation 50 times per minute in the presence of serum-free medium (SFM), normolipemic serum (NLS), or hyperlipemic serum (HLS). Incorporation of 14C-proline into proline and into hydroxyproline was taken as a measure of protein and collagen synthesis. When cells were grown in plastic Petri dishes, exposure to NLS or HLS increased both protein and collagen production to the same extent compared to synthesis in SFM (1.7 times for NLS and 1.6 times for HLS; p less than 0.001 compared to SFM). For cells grown on stationary elastin membranes, NLS and HLS also increased protein and collagen synthesis compared to SFM. The effect of NLS was 1.35 times that of HLS for protein and 1.43 times greater for collagen (p less than 0.03). Cyclic stretching in SFM doubled synthesis for both protein (p less than 0.002) and collagen (p less than 0.002) compared to stationary controls, but had no effect on synthesis in NLS. In HLS, however, cyclic stretching elevated synthesis to the same level as was found in NLS (p less than 0.003). We conclude that the relative inhibition of synthesis on stationary membranes by HLS was not due to a toxic effect, since HLS increased synthesis both in Petri dishes and on elastin membranes, and the amplifying effect of cyclic stretching in HLS was similar to that seen in SFM.(ABSTRACT TRUNCATED AT 250 WORDS)

N-Acetyl-β-hexosaminidase B deficiency in cultured fibroblasts from a patient with progressive motor neuron disease

A patient with a 20-year history of progressive motor neuron disease was previously found to have profoundly low levels of N-acetyl-beta-hexosaminidase (Hex) in serum and leukocytes; Hex activity in cultured skin fibroblasts was in the low normal range. By thermal inactivation and cellulose acetate electrophoresis, the residual activity appeared to be Hex A. In the present study, the residual activity in cultured skin fibroblasts was further characterized as Hex A by thermal inactivation at reduced temperatures and ion exchange chromatography; no evidence was obtained for a diffusible inhibitor of Hex activity. After labeling with [3H]leucine, immunoprecipitation with polyclonal antibody to Hex B, and SDS-polyacrylamide gel electrophoresis, both alpha and beta polypeptide chains were visualized, confirming the presence of Hex A. The results suggest that, in the patients fibroblasts, a defect in beta-chain synthesis or processing precludes the self-association of beta-chains to form Hex B, but does not prevent the association of alpha- and beta-chains to form Hex A.

Rapid degradation of steroid sulfatase in multiple sulfatase deficiency

Pulse labeling followed by SDS-PAGE electrophoresis of immunoprecipitated [35S]methionine-labeled steroid sulfatase (STS) gave a single band of molecular weight 65,000 daltons. After a chase period of 18 hours the material appeared as molecular weight approximately 64,000. No labeled STS could be detected in fibroblasts from individuals with STS deficient X-linked ichthyosis. Pulse-chase labeling of normal and multiple sulfatase deficiency (MSD) fibroblasts showed a normal rate of synthesis of STS in MSD during a 3 hour pulse but during the chase the STS of MSD cells disappeared with a half-life of 4 to 6 hours until approximately 25% of the material remained after 24 hr. STS of normal cells had a half-life of 6 days. The material produced in MSD cells had the same molecular size as normal and had the same amount of endoglycosidase sensitive carbohydrate as normal. The defect in MSD thus seems to result in degradation after the addition of N-linked oligosaccharides.

Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin.

In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed. This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42%. The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure. Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3). In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained. During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium. Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein. The Ka fo. L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors. Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4. Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks. These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS)