Molecular organization of the human cathepsin D gene.

A 16-kb fragment of human DNA containing the cathepsin D (CATD) gene was isolated. Nucleotide sequencing, primer extension, protection from mung bean nuclease, and promoter activity assays were used to characterize the gene. The transcribed portion of the gene is about 11,000 bp and is organized into 9 exons analogous with the human pepsinogen A gene. Human pepsinogen A and CATD proteins have 42% sequence identity, while the two cDNAs are 55.7% identical. The positions of the splice junctions are fully conserved in these two genes. The noncoding sequences of the two genes are dissimilar. We report the nucleotide sequence of an Eco RI-Bam HI fragment that contains the transcription initiation site. The promoter region contains no TATA and CCAAT boxes, but five potential Sp1 binding sites (one of them in the first intron) and four AP-2 binding sites (two of them in the first intron). In COS-1 cells, the region containing the three proximal Sp1 sites possesses the bulk of the promoter activity of the 5'-flanking sequence. The transcription start site of the CATD gene is localized within a CpG cluster. In the interval -390 through +450, the content of CpG is 5.8 times above the average throughout the human genome.

UGA is translated as cysteine in pheromone 3 of Euplotes octocarinatus.

Pheromone 3 mRNA of the ciliate Euplotes octocarinatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. No UAG codon was found. In connection with results reported for other ciliates, this suggests that the role of the classic termination codons had not yet been established when the ciliates started to diverge from other eukaryotes.

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.

Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor.

Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in lambda gt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids (Mr = 30,993), and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (Mr = 18,299) and C-terminal (Mr less than or equal to 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12.

Influence of effectors of the complex-type-oligosaccharide biosynthesis on the formation of proteokeratan sulfate in bovine cornea.

The structural similarity of the inner core of complex-type prosthetic oligosaccharides of N-asparagine glycoproteins and of the linkage region between the polysaccharide part and the protein chain of cornea proteokeratan sulfate makes their biosynthesis via a common route an attractive hypothesis. To test this, a tissue culture system was established to determine the rate of proteokeratan sulfate biosynthesis in bovine cornea and to measure the influence of several effectors of the dolichol pathway on this rate. Addition of dolichyl phosphate enhanced the formation of proteokeratan sulfate. Tunicamycin, 2-deoxy-D-glucose, bromoconduritol and deoxynojirimycin inhibited this process. Swainsonine probably led to the formation of a keratan sulfate with hybrid structure. The results support that the linkage region of cornea proteokeratan sulfate is synthesized via the assembly of a glucosylated dolichyl pyrophosphoryl oligosaccharide, its transfer to protein and subsequent processing by glycosidases.

beta-D-Mannosidase from human placenta: properties and partial purification.

beta-D-Mannosidase was partially purified (108-fold) from human placenta by ammonium sulfate precipitation, Concanavalin A-Sepharose affinity chromatography, gel filtration and hydroxylapatite chromatography. Further attempts to purify the enzyme by several conventional methods failed due to loss of activity. The stability of beta-mannosidase, the effect of several compounds on its activity and some physico-chemical and kinetic parameters were investigated.

The carbohydrate-protein-binding region in proteokeratan sulfate from bovine cornea is not sulfated.

Peptidokeratan sulfate from bovine cornea was degraded without prior desulfation by hydrazinolysis and nitrous acid deamination and the products were reduced and labelled with NaB3H4. The chain disaccharide units gave, as expected, galactosyl-2,5-anhydromannitol disaccharides with no, one and two sulfate groups, respectively. Desulfation during the degradation procedure was less than 25%. The fraction of the degradation products containing the binding region was identified by its mannose content and size, further separated by mannose-specific affinity chromatography and high-voltage electrophoresis and analysed. 94% of its mannose was found in uncharged oligosaccharides. This indicates that the binding region oligosaccharide bears no sulfate groups.

The carbohydrate-protein binding region in keratan sulfate from bovine cornea, structure of a major binding region oligosaccharide.

Peptido-keratan sulfate from bovine cornea was degraded by a combination of desulfation, hydrazinolysis, nitrous acid deamination and NaB3H4 reduction. The tetrasaccharide fraction obtained by gel filtration was studied by degradation with specific exoglycosidases and methylation analysis. The existence of two different binding region oligosaccharide structures was established: The first structure contains one terminal fucose, two mannosine residues and N-acetylglucosamine at the reducing end. In the second structure one N-acetylglucosamine is bound to the protein backbone and substituted with branched 3,6-di-O-alpha-mannosyl-beta-mannose. Both terminal alpha-mannosine residues bear keratan sulfate chains in the 2-position.

The carbohydrate-protein binding region in keratan sulfate from bovine cornea. I. Isolation and partial characterization.

We present a defined chemical method for the isolation of the oligosaccharide linking the protein to the disaccharide units of keratan sulfate from bovine corneas. 1) Hydrazinolysis removes the amino acids from the peptido keratan sulfate, and leads to deacetylation of the glucosamine residues but does not split off the sulfate groups. 2) NaB3H4 reduction selectively labels the reducing terminal glucosamine of the chain by converting it to [3H]glucosaminitol. 3) Nitrous acid deamination splits the glycosidic bonds of glucosamine and converts this sugar into 2,5-anhydromannose but also leads to several derivatives of the free terminal [3H]glucosaminitol. 4) Na12CN treatment stabilizes the reactive 2,5-anhydromannose and the terminal compounds containing aldehyde groups in a cyanhydrin reaction. 5) The oligosaccharide structure between the glucosamine residue at the reducing end and the first glucosamine of the disaccharide chain is not degraded by this procedure and is obtained intact and in labelled form. The data so far obtained on this part of the cornea keratan sulfate show that it is heterogeneous and contains besides the terminal glucosamine, mannose and fucose in a similar ratio as found in undegraded keratan sulfate. The predominant compound probably contains three neutral sugar residues.

Endocytosis of lysosomal enzymes by non-parenchymal rat liver cells. Comparative study of lysosomal-enzyme uptake by hepatocytes and non-parenchymal liver cells.

Cultured non-parenchymal rat liver cells internalize human urine alpha-N-acetylglucosaminidase, human skin beta-N-acetylglucosaminidase and pig kidney alpha-mannosidase. Different heat-stabilities of endocytosed and endogenous alpha-mannosidase activity provided indirect evidence that the increase in intracellular activity resulted from uptake. The high efficiency and the saturation kinetics of uptake indicated that these enzymes become internalized by adsorptive endocytosis. Competition experiments with glycoproteins bearing known carbohydrates at their non-reducing terminals, with mannans, methyl glycosides and monosaccharides, established that the uptake of these three lysosomal enzymes is mediated by the binding to cell-surface receptors that recognize mannose and N-acetylglucosamine residues. The decreased uptake after treatment of these enzymes with either beta-N-acetylglucosaminidase or alpha-mannosidase was in accordance with the results of the inhibition experiments. Removal of oligosaccharides of the high-mannose type by treatment with endoglucosaminidase H inhibited uptake almost completely, suggesting that the sugars recognized by cell-surface receptors of non-parenchymal liver cells are located in the outer core of these oligosaccharides. A comparison of the uptake of these three lysosomal enzymes by parenchymal and non-parenchymal rat liver cells indicates that infused alpha-N-acetylglucosaminidase is taken up preferentially by hepatocytes, whereas alpha-mannosidase and beta-N-acetylglucosaminidase are localized predominantly in non-parenchymal rat liver cells.

Epithelial rat liver cells have cell surface receptors recognizing a phosphorylated carbohydrate on lysosomal enzymes.

Receptor-mediated endocytosis of alpha-N-acetylglucosaminidase by cultured epithelial rat liver cells is inhibited by mannose, L-fucose and most effectively by mannose 6-phosphate. Endocytosis of alpha-N-acetylglucosaminidase is lost after treatment of the enzyme with alkaline phosphatase. These findings indicate that epithelial rat liver cells possess cell surface receptors that recognize a phosphorylated carbohydrate on alpha-N-acetylglucosaminidase, as was previously reported for cell surface receptors of human skin fibroblasts. Inhibition of alpha-mannosidase endocytosis by epithelial rat liver cells in the presence of mannose 6-phosphate and loss of enzyme endocytosis after treatment with alkaline phosphatase suggest that this enzyme is recognized by the same receptor.

Amount and properties of uptake forms in preparations of alpha-mannosidase from pig kidney.

alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from pig kidney has been shown to exist in multiple forms differing in their capability to be endocytosed by alpha-mannosidase deficient cultured cells. A method is presented to evaluate the amount of "uptake" forms in different preparations of the enzyme. Preparations with different rates of uptake were shown to contain different amount of "uptake" forms and "non-uptake" forms. The content of "uptake" forms in a preparation was identical with that of enzyme molecules bearing a phosphorylated carbohydrate group necessary for the recognition by cell surface receptors.

Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety.

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl alpha-d-mannoside, alpha-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for beta-glucuronidase from human urine. The inhibition of alpha-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl alpha-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A.74, 2026-2030]. Endocytosis of ;low-uptake' forms of alpha-N-acetylglucosaminidase and alpha-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that ;low-uptake' forms are either contaminated with ;high-uptake' forms or are internalized via the same route as ;high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver beta-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.

Differences in intracellular corrective activity in cultured mannosidosis cells of acidic alpha-mannosidases from various sources.

The ability of acidic alpha-mannosidases from pig kidney, bovine liver, human urine and placenta, and jack bean to correct the deranged metabolism of cultured skin fibroblasts from manosidosis patients is studied. One cause for the different corrective abilities are, as shown for other systems, the different rates of endocytosis of the enzyme forms into the cells. Besides this, it is demonstrated for the first time, that there are differences in the intracellular corrective activity of the internalized enzymes, which can be explained by different specificities of the enzymes against the storage material.

Storage of mannose-containing material in cultured human mannosidosis cells and metabolic correction by pig kidney alpha-mannosidase.

Skin fibroblasts from healthy individuals and a mannosidosis patient were cultured in the presence of [2-3H] mannose and the cell homogenates were fractionated by trichloroacetic acid precipitation into a precipitable and a non-precipitable portion. In uptake as well as in chase experiments the precipitable fractions show no significant difference in their content of radioactivity, while an increased level of radioactivity is found in the non-precipitable fraction of mannosidosis cells. This higher radioactivity content is due to a higher mannose content and is caused by a slower degradation of this fraction. The differences between the metabolisms of the two cell lines can be expressed by the ratio of radioactivity in the non-precipitable and the precipitable fractions. This value is about three times higher for mannosidosis than for control cells. Pig kidney alpha-mannosidase is taken up by both cell lines and is able to correct the impaired degradation of the non-precipitable material in mannosidosis cells, as shown by the normalization of the above defined ratio of radioactivity for this cell type.