Kurt von Figura

Intracellular receptor sorting during endocytosis: Comparative immunoelectron microscopy of multiple receptors in rat liver

Using double-label quantitative immunoelectron microscopy on ultrathin cryosections of rat liver, we have compared the endocytotic pathways of the receptors for asialoglycoprotein (ASGP-R), mannose-6-phosphate ligands (MP-R), and polymeric IgA (IgA-R). All three were found within the Golgi complex, along the entire plasma membrane, in coated pits and vesicles, and within a compartment of uncoupling of receptors and ligand ( CURL ). The receptors occurred randomly at the cell surface, in coated pits and vesicles. Within CURL tubules ASGP-R and MP-R were colocalized , but IgA-R and ASGP-R displayed dramatic microheterogeneity. Thus, in addition to its role in uncoupling and sorting recycling receptor from ligand, CURL serves as a compartment to segregate recycling receptor (e.g. ASGP-R) from receptor involved in transcytosis (e.g. IgA-R).

Enzymatic phosphorylation of lysosomal enzymes in the presence of UDP-N-acetylglucosamine. Absence of the activity in l-cell fibroblasts

Abstract 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- 32 P]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-[ 32 P]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.

Deficiency of UDP-N-acetylglucosamine: Lysosomal enzyme N-acetylglucosamine-1-phosphotransferase in organs of I-cell patients

Abstract A N-acetylglucosamine-1-phosphotransferase is involved in synthesis of a common phosphorylated recognition marker in lysosomal enzymes. Absence of this enzyme in liver, spleen, kidney and brain of two patients with I-cell disease is now reported. In these organs activities of lysosomal enzymes are close to normal. In contrast, in fibroblasts the absence of N-acetylglucosamine-1-phosphotransferase and of the common recognition marker are known to result in a severe intracellular deficiency of lysosomal enzymes. It is proposed that in certain organs the transport of lysosomal enzymes into lysosomes is mediated by alternative systems, which recognize structural features other than the phosphorylated recognition marker.

Primary cultures of rat hepatocytes synthesize fibronectin.

Summary Fibronectin was detected by indirect immunofluorescence on primary cultures of rat hepatocytes maintained in the presence or absence of fetal calf serum. [ 14 C]-Fibronectin synthesized in the presence of [ 14 C]-glycine was isolated by immunoprecipitation and visualized by fluorography.

The Sanfilippo B corrective factor: A N-acetyl-α-D-glucosaminidase

Summary Fibroblasts from patients with Sanfilippos disease fall into two groups — A and B, each deficient in a specific protein factor which is required for normal degradation of sulfated mucopolysaccharide. From normal human urine the factor deficient in the B subgroup was copurified with N-acetyl-α-D-glucosaminidase by ammonium sulfate precipitation and successive chromatography on Sephadex G-200 and carboxymethyl cellulose. Both the factor and the enzyme had the same relative mobility in three different polyacrylamide gel electrophoresis systems at several gel concentrations. Since Sanfilippo B fibroblasts were found to be strikingly deficient in N-acetyl-α-D-glucosaminidase activity, this inactivity is proposed to be the basic defect in Sanfilippo B disease.

Partial purification and characterization of a heparan sulfate specific endoglucuronidase

Abstract A heparan sulfate degrading endoglycosidase was partially purified from human placenta. The analysis of the reducing end groups liberated by the endoglycosidase identified this enzyme as an endoglucuronidase. This endoglucuronidase is most active at pH values below pH 6.5 and appears to be a glycoprotein. The enzyme is specific for heparan sulfate and depolymerizes its substrate to oligosaccharides.

Evidence for degradation of heparan sulfate by endoglycosidases: glucosamine and hexuronic acid are reducing terminals of intracellular heparan sulfate from human skin fibroblasts.

Summary Intracellular heparan sulfate was isolated from fibroblasts cultured from the skin of patients with Sanfilippo disease type B (mucopolysaccharidosis III B) and with Scheies diseae (mucopolysaccharidosis I S). In both diseases, the stored heparan sulfate exhibits an average molecular size of about on sixth of that of extracellular heparan sulfate. Upon reduction with [3H]NaBH4 [3H]glucosaminitol and [3H]aldonic acid were identified as the [3H]sugar derivatives at the reducing terminal of intracellular heparan sulfate. These results suggest the participation of an endoglucosaminidase and an endohexuronidase in the degradation of heparan sulfate.

Localization of sulfated glycosaminoglycans within cell nuclei by high-resolution autoradiography.

Abstract Skin fibroblasts cultured from patients with Sanfilippos disease type B and Hurlers disease, preincubated with [35S]sulfate, were subjected to high resolution autoradiography. A 4-day pulse followed by a 24 h chase resulted in a labelling of 61% of the cell nuclei by silver grains, thus indicating the existence of ethanol-insoluble [35S]sulfate-containing material within the nuclei, especially within their chromatin-rich peripheral zone. A computer-calculated statistical evaluation of the autoradiographic results showed that the silver grains overlying the cell nuclei originated from [35S]radioactivity within the nuclei and not from an overall background or cross fire effects of cytoplasmic radiation sources. Chemical analyses of chloroform/methanol extracts and pronase digests of the [35S]labelled cells provided evidence that neither [35S]sulfatides nor [35S]glycopeptides contribute substantially to the cellular [35S]radioactivity. The results strongly suggest the association of sulfated glycosaminoglycans with cell nuclei.

Immunoelectron-microscopic study on the location of fibronectin in human fibroblast cultures

By immunoelectron-microscopic techniques and the recently developed carbon-film cytochemistry it was possible to localize fibronectin or fibronectin-containing material on cultured human skin fibroblasts. In carbon-film replicas of cell surfaces this material is arranged in the form of a fibrous network. In ultrathin sections, this network appears as rather bulky patches on the cell surfaces. In addition fibronectin is often observed in the interspaces between neighboring cells. This finding supports the assumption that fibronectin is involved in the mechanisms of cell-cell contact, cell-substrate adhesion, and cell recognition.

Physical properties and biological activities of two forms of α-N-acetylglucosaminidase from bovine spleen

Abstract α - N -Acetylglucosaminidase (2-acetamido-2-deoxy-α- d -glucoside acetamido-deoxyglucohydrolase, EC 3.2.1.50) was purified 108-fold from bovine spleen homogenates and could be separated into two interconvertible forms (I and II) by gel filtration and disc electrophoresis, whereas separation was not possible by DEAE-cellulose chromatography or isoelectric focusing. The two forms have a molecular weight ratio of 2:1 and display identical enzyme kinetics and the same biological activity in the correction of the altered heparan sulfate catabolism of cultured human Sanfilippo B fibroblasts. The lower corrective activity of the bovine α - N -acetylglucosaminidase as compared with that of the human urinary enzyme is caused by a lower rate of uptake into the fibroblasts.