Identification of a mutation in the structural alpha-L-fucosidase gene in fucosidosis.

Fucosidosis is an autosomal recessive lysosomal storage disorder characterized by progressive neurological deterioration and mental retardation. The disease results from deficient activity of alpha-L-fucosidase (E.C.3.2.1.51), a lysosomal enzyme that hydrolyzes fucose from fucoglycoconjugates. In an attempt to identify the mutation(s) that result(s) in fucosidosis, we performed Southern blot analysis of the structural gene encoding alpha-L-fucosidase (FUCA 1) in 23 patients affected with fucosidosis. In five patients Southern blot analysis showed obliteration of an EcoRI restriction site in the open reading frame of FUCA 1 encoding mature alpha-L-fucosidase. This abnormality was not observed in 80 controls, and it may be the basic defect responsible for fucosidosis in these patients. Both patients with the severe type I form of fucosidosis and patients with the less severe type II were shown to be homozygous for this presumed mutation. In the remaining 18 patients the EcoRI site obliteration, major-gene deletions, or insertions were not detected. This suggests that at least two different mutations are involved in fucosidosis. The heterogeneity found at the DNA level was not present at the protein level, as all fucosidosis patients investigated had low fucosidase protein (less than 6% of normal) and negligible fucosidase activity in fibroblasts and lymphoblastoid cell lines.     

Molecular biology of the alpha-L-fucosidase gene and fucosidosis

Human alpha-L-fucosidase, a lysosomal enzyme, hydrolyzes alpha-L-fucose from glycolipids and glycoproteins. Its activity is deficient in human fucosidosis an autosomal recessive disease. In order to understand the molecular basis of this lysosomal storage disorder we have cloned several cDNAs coding for human alpha-L-fucosidase from a human hepatoma and a human liver cDNA library constructed in lambda gt11. Compiling the cDNA sequences of these clones we have identified 1,829 base pairs (bp) encoding human alpha-L-fucosidase. This includes an open reading frame of 1,172 bp, a consensus polyadenylation signal AAT AAA and a poly(A)+ tail. The sequence is incomplete at the 5'-end, and clones encoding the amino terminus of the native protein, the propeptide and leader signal have not yet been isolated. The open reading frame encodes for 390 amino acids with a calculated Mr of 45,557. This represents 78-95% of the mature processed alpha-L-fucosidase. The availability of these cDNA clones has enabled us to map the structural gene for alpha-L-fucosidase to chromosome 1p34.1-1p36.1 by Southern blot analysis of DNA from human-rodent somatic cell hybrids and by in situ hybridization. Furthermore, a Pvu II restriction fragment length polymorphism (RFLP) has been identified at the human alpha-L-fucosidase gene locus. Analysis of mRNA by Northern blotting gives a major species of 2.25 kb. In 4 patients with fucosidosis no mRNA signal was detected and Western blots gave no immunoreactive enzyme. Southern blotting after Eco RI digestion in two fucosidosis families revealed a banding abnormality (extra 6-kb band).(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation generating a stop codon in the alpha-L-fucosidase gene of a fucosidosis patient.

Fucosidosis is an autosomal recessive, lysosomal storage disease featured by deficient activity of alpha-L-fucosidase. Lymphoid cell lines from a fucosidosis patient (JT) and a healthy individual (control) contained alpha-L-fucosidase mRNA of the same size, 2.3 Kb, as determined by Northern blot analysis. cDNA was prepared from alpha-L-fucosidase mRNA of JT and control cells and each cDNA was amplified by the polymerase chain reaction. Direct DNA sequencing of the amplified products revealed a single mutation in JT, a G1141-->T transition. This changed the codon (GAA) for Glu-375 to a stop codon (UAA). Amplification and sequencing of the area containing the G1141-->T transition in genomic DNA of JT and control cells demonstrated that the mutation was homozygous in JT. Analysis of cDNA and genomic DNA derived from lymphoid cells of mother JT revealed her to be heterozygous (G and T) at position 1141. The G1141-->T mutation is probably responsible for disease in JT.     

Molecular defect in processing alpha-fucosidase in fucosidosis

In normal human skin fibroblasts, an enzymatically active 53,000-dalton form of alpha-fucosidase is processed to a 50,000-dalton mature form. Endoglycosidase-H treatment of [35S]methionine pulse-chase labelled material immunoprecipated with a polyclonal antibody to alpha-L-fucosidase (Andrews-Smith & Alhadeff, Biochim. Biophys. Acta 715: 90-96 (1982)) indicated the removal of a single N-linked oligosaccharide unit from both precursor and mature form of alpha-L-fucosidase. Tunicamycin pretreatment of normal fibroblasts indicated that no other N-linked oligosaccharide units were present. Studies on fibroblasts from patients with less than 5% of normal alpha-L-fucosidase activity (fucosidosis) showed 8 of 11 patients synthesized no detectable alpha-fucosidase protein whereas 2 synthesized normal amounts of 53,000 dalton precursor, none of the mature 50,000 dalton form was detectable and one contained small amounts of cross-reacting material. This is the first evidence for processing of alpha-L-fucosidase in cells and the first precise evidence of a molecular defect in fucosidosis.     

Restriction analysis of the structural α-L-fucosidase gene and its linkage to fucosidosis

Human alpha-L-fucosidase is a lysosomal enzyme responsible for hydrolysis of alpha-L-fucoside linkages in fucoglycoconjugates. A single gene, FUCA 1, located on chromosome 1p34.1-1p36.1 encodes for alpha-L-fucosidase activity. To gain insight into the nature of the molecular defects leading to fucosidosis, we have characterized the genomic structure of FUCA 1. Restriction-endonuclease analysis suggests that at least seven exons dispersed over 22 kb are present in genomic FUCA 1. Two restriction-fragment-length polymorphisms (RFLPs) have been identified in the Caucasian population. The PvuII and BglI RFLPs each have two codominant alleles in Hardy-Weinberg equilibrium. Allele frequencies for the PvuII RFLP are .70/.30, and those for the BglI RFLP .63/.37. Both RFLPs are in strong linkage disequilibrium with each other, with a correlation coefficient of .94. The polymorphism information content (PIC) of the combined DNA markers is .38, high enough to be useful in the prenatal diagnosis of fucosidosis. The combined lod score for linkage between the fucosidosis mutation and FUCA 1 markers in two families was significant at a recombination fraction of 0. This suggests that the fucosidosis mutation resides in FUCA 1.     

Metabolic correction of fucosidosis lymphoid cells by galaptin-alpha-L-fucosidase conjugates

To determine if isolated galaptin, an endogenous galactoside-binding lectin, could serve as a transport vehicle of therapeutic agents to cells, galaptin and alpha-L-fucosidase were coupled using glutaraldehyde. The conjugates were incubated with alpha-L-fucosidase-deficient, EBV-immortalized lymphoid cells from a fucosidosis patient. Conjugates were effectively bound and internalized by the cells in a lactose inhibitable manner. Internalization of conjugate resulted in the reduced accumulation of alpha-L-fucosyl-N-acetylglucosaminylasparagine, a glycopeptide that accumulates in cells of fucosidosis patients, to levels found in lymphoid cells from a healthy individual. Thus, galaptin-alpha-L-fucosidase conjugates may be useful for enzyme replacement therapy of fucosidosis. The concept of using galaptin as a transport vehicle may be applied to the delivery of other compounds to cells bearing galaptin receptors.     

Canine alpha-L-fucosidase in relation to the enzymic defect and storage products in canine fucosidosis.

Canine liver alpha-L-fucosidase was purified to apparent homogeneity by affinity chromatography on agarose-epsilon-aminohexanoyl-fucopyranosylamine. It is composed of multiple forms of a common active subunit of 45-50 kDa, which can aggregate in different combinations to form polymers, predominantly dimers. Antiserum was raised against the purified enzyme. There is negligible residual alpha-L-fucosidase in the tissues of English springer spaniels with the lysosomal storage disease fucosidosis. Although no alpha-L-fucosidase protein was detected by Western blotting or by the purification procedure in the affected tissues, some enzymically inactive cross-reacting material was detected in both normal and affected tissues. This suggests that another protein without alpha-L-fucosidase activity was co-purified with the enzyme. Dog liver alpha-L-fucosidase was precipitated by goat anti-(human liver alpha-L-fucosidase) IgG, indicating homology between the enzymes in the two species. Two purified storage products isolated from the brain of a dog with fucosidosis were used as natural substrates for various preparations of canine liver alpha-L-fucosidase. Analysis of the digestion mixtures by t.l.c. and fast-atom-bombardment mass spectrometry suggests that canine alpha-L-fucosidase acts preferentially on the alpha-(1-3)-linked fucose at the non-reducing end and that removal of alpha-(1-6)-linked asparagine-linked N-acetylglucosamine is rate-limiting in the lysosomal catabolism of fucosylated N-linked glycans.     

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.     

Biochemical and genetic studies of plasma and leukocyte α-L-fucosidase

Deficiency of alpha-L-fucosidase in plasma and leukocytes has been demonstrated in three patients affected with fucosidosis. The measurement of plasma fucosidase activity alone is of little diagnostic value. Several normal individuals were found to have extremely low plasma alpha-L-fucosidase activity, but normal activity in leukocyte preparations. The low plasma enzyme activity exhibited by clinically normal individuals appears to be an inherited characteristic. The plasma enzyme was found to be different from that of leukocytes in terms of electrophoretic mobility.     

Human alpha-L-fucosidase: complete coding sequence from cDNA clones

The human lysosomal storage disorder fucosidosis results from the deficiency of alpha-L-fucosidase, a lysosomal enzyme essential for the catabolism of oligosaccharides containing alpha-L-fucosides. cDNA clones coding for human alpha-L-fucosidase have been isolated from lambda gt10 and lambda gt11 cDNA libraries derived from human liver, placenta and colon. Compilation of cDNA sequences results in a nucleotide sequence of 2053 base pairs encoding alpha-L-fucosidase. The sequence contains an open reading frame of 461 amino acids beginning with the first in-frame methionine and includes 439 amino acids which comprise the mature protein in addition to a hydrophobic signal peptide sequence of 22 amino acids.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

Isolation and characterization of two families of satellite DNA with repetitive units of 135 bp and 2.5 kb in the ant Monomorium subopacum (Hymenoptera, Formicidae)

Analyzing the satellite DNA in the ant species Monomorium subopacum we found two unrelated families of satellite DNA. Because these satellite DNA families were isolated using the two enzymes HaeIII and EcoRI we called the two families HaeIII and EcoRI family, respectively. The HaeIII family proved to be organized in a 135-bp basic unit repeat, the EcoRI family in a 2.5-kb basic unit repeat. The latter represents perhaps the longest satellite DNA isolated up to now in insects. The HaeIII family apparently comprises about 10% of the total genomic DNA whereas the EcoRI family represents only about 1-2%. A comparative analysis of the two satellite DNA sequences showed no homology between the two families although both sequences possessed long A and T stretches. Eight of the 34 chromosomes showed hybridization with the HaeIII family and hybridization signals are visible in six chromosomes with the EcoRI family. Analysis of the electrophoretic mobility of satellite DNA on non-denaturing polyacrylamide showed that the HaeIII family is only slightly curved. However, the unit of the EcoRI satellite DNA family has curvature, especially the first 1000 bp of the monomeric repeat, in which this DNA is AT rich and has numerous A and T stretches. There are also internal inverted subrepeats in each family. The sequences of satellite DNA families found in Monomorium subopacum are different from the sequences of other satellite DNAs cloned in insects, including other species of ants.     

The enzymic defect and storage products in canine fucosidosis.

A marked deficiency of alpha-L-fucosidase and the accumulation of fucose-containing glycoasparagines were found in the brains of two English Springer spaniels suffering from a progressive nervous disorder. Both forms of alpha-L-fucosidase in normal brain, which are separable by ion-exchange chromatography, are absent from the affected animals. The storage products were characterized by t.l.c., gel filtration, g.l.c. and fast-atom-bombardment mass spectrometry. The postulated structures of the main components are: (formula; see text) The enzymic defect and nature of storage products justify designation of this disorder as canine fucosidosis.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

Molecular cloning of genomic DNA segments partially coding for human thymidylate synthase from the mouse cell transformant

Mouse cells deficient in the enzyme thymidylate synthase [TS; EC 2.1.1.45] were serially transformed with human DNA to yield primary and secondary transformants which produced human TS [Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., & Seno, T. (1983) J. Biol. Chem. 258, 48-53]. Southern blot hybridization of their genomic DNA showed that six secondary transformants examined contained in common a 5.5 kb EcoRI fragment hybridized with a human Alu sequence. From the secondary transformant genomic library constructed with phage lambda Charon 4A, two recombinant phage clones carrying Alu sequences were isolated. Restriction endonuclease mapping revealed that the insert DNAs of the two phage clones overlapped and covered a region of 19 kb in total. Within this region at least six Alu sequences were located. A 2.0 kb DNA fragment, prepared from an EcoRI fragment subcloned in plasmid pBR322 and free of Alu sequences, hybridized to a single band on RNA blots of primary and secondary transformant poly(A)+ RNA, but not to RNA of mouse wild-type and recipient cell lines. The relative amount of the presumed human TS mRNA was linearly correlated with the relative activity of human TS in various types of mouse transformant cells. These results indicate that these two phage clones contain genomic DNA sequences encoding human TS.     

Characterization of a New Type of Human Papillomavirus That Causes Skin Warts

A human papillomavirus (HPV) was isolated from the lesions of a patient (ML) bearing numerous hand common warts. This virus was compared with the well-characterized HPV found in typical plantar warts (plantar HPV). ML and plantar HPV DNAs have similar molecular weights (5.26 x 10(6) and 5.23 x 10(6), respectively) but were shown to be different by restriction enzyme analysis. When the cleavage products of both DNAs by endonuclease EcoRI, BamI, HpaI, or Hind were analyzed by electron microscopy, one, two, one, and four fragments were detected for ML HPV DNA instead of the two, one, two, and six fragments, respectively, detected for plantar HPV DNA. In contrast to plantar HPV DNA, a high proportion of ML HPV DNA molecules were resistant to these restriction enzymes. Most, if not all, of the molecules were either resistant to BamI and sensitive to EcoRI or sensitive to BamI and resistant to EcoRI. After denaturation and renaturation of the cleavage products of ML HPV DNA by a mixture of the two enzymes, the circular "heteroduplexes" formed showed one to three heterology loops corresponding to about 4 to 8% of the genome length. No sequence homology was detected between ML and plantar HPV DNAs by cRNA-DNA filter hybridization, by measuring the reassociation kinetics of an iodinated plantar HPV DNA in the presence of a 25-fold excess of ML HPV DNA, or by the heteroduplex technique. The two viruses had distinct electrophoretic polypeptide patterns and showed no antigenic cross-reaction by immunodiffusion or immunofluorescence techniques. Preliminary cRNA-DNA hybridization experiments, using viral DNAs from single or pooled plantar or hand warts, suggest that hand common warts are associated with viruses similar or related to ML HPV. The existence of at least two distinct types of HPVs that cause skin warts was demonstrated; they were provisionally called HPV type 1 and HPV type 2, with plantar HPV and ML HPV as prototypical viruses, respectively.     

Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene

Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of a cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage lambda was screened using the chicken cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show that both genes are transcribed at about the same level when yeasts are grown on glucose media.     

A new type of EcoRI polymorphism of the human ribosomal DNA repeating unit revealed by analysis of cloned DNA fragments

Several clones of rDNA have been isolated from an adult human liver DNA Charon 4A library by using cDNA probes synthesized from human 18S and 28S rRNA. The insert of one recombinant Charon 4A clone contained, besides the already known 5.7-kb EcoRI fragment of rDNA, comprising the major portion of the 18S rRNA gene and all the external transcribed spacer (ETS), a previously unidentified EcoRI fragment of rDNA of 8.5 kb in size. DNA transfer hybridization experiments utilizing EcoRI digests of the human DNA used to construct the library and of another human DNA showed the presence of the 8.5-kb EcoRI fragment in a minority of the rDNA repeats on the 5'-end side of the 5.7-kb fragment, thus defining a hitherto unidentified type of EcoRI polymorphism of these repeats.