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A common polymorphism of human alpha-L-fucosidase consists of three phenotypes (Fu 1, Fu 2, and Fu 2-1) assigned by isoelectric focusing. The phenotypes are determined by two codominant alleles (Fu1 and Fu2). Isozymes with the Fu 2 phenotype have more basic pI than Fu 1, while Fu 2-1 is a mixture of Fu 2 and Fu 1. Recently, a missense mutation (A860-->G) in the alpha-L-fucosidase gene was described that did not affect alpha-L-fucosidase activity. The mutation causes the substitution of Arg (pKaGuan = 12.5) for Gln-281, which has no ionizable side chain. Isoelectric focusing profiles of extracts of COS-1 cells transfected with wild-type or mutant alpha-L-fucosidase cDNAs had phenotypes of Fu 1 and Fu 2, respectively. Next, 20 human lymphoid cell lines were examined for the occurrence of the A860-->G mutation and expression of the Fu 1, Fu 2, and Fu 2-1 phenotypes. Eight lines with Fu 2 were homozygous for the A860-->G mutation; six lines with Fu 1 were homozygous for the normal nucleotide (A860); and six lines with Fu 2-1 were heterozygous. Thus, the A860-->G mutation is the molecular basis for the protein phenotypes and the Fu1 and Fu2 alleles. The normal nucleotide (A860) is responsible for Fu1 and the A860-->G mutation for Fu2.

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from absence of alpha-L-fucosidase activity. Lymphoid cell lines from two siblings with fucosidosis and a healthy individual (control) had alpha-L-fucosidase mRNA of normal size (2.3 kb) but the level of alpha-L-fucosidase mRNA in the patients' cells was reduced. cDNA was prepared and amplified from alpha-L-fucosidase mRNA of lymphoid cells of the patients, their carrier parents, and the control. Direct DNA sequencing demonstrated three mutations in the fucosidosis family. One mutation, C1282-->T, changed the codon (CAA) for Gln-422 to a stop codon (UAA). This mutation was heterozygous (C and T) in the patients and their father and independently confirms an earlier report (J. Mol. Neurosci. (1989) 1, 177). Another mutation, C247-->T, changed the codon (CAG) for Gln-77 to a stop codon (UAG) and was heterozygous (C and T) in the patients and their mother. The third mutation, A860-->G, changed the codon CAG for Gln-281 to the codon (CGG) for Arg and was heterozygous (A and G) in the patients but homozygous in their father. alpha-L-Fucosidase activity in cells of the father was 37% of controls indicating that homozygosity of the A860-->G mutation did not cause an absence of alpha-L-fucosidase activity and fucosidosis. This mutation probably results in a normal polymorphic variant of alpha-L-fucosidase. It is proposed that the combination of the C247-->T mutation on the maternal allele of the alpha-L-fucosidase gene and the C1282-->T mutation on the paternal allele caused fucosidosis in the patients.

Mannose 6-phosphate is a recognition marker used by many newly made acid hydrolases for their transport to lysosomes. Previously, we investigated the incorporation of 32Pi into alpha-L-fucosidase of lymphoid cell lines from a healthy individual (control) and an I-cell disease patient [DiCioccio and Miller, Glycobiology, 1, 595-604 (1991)]. Phosphoserine was found in immunoprecipitable alpha-L-fucosidase of both control and I-cell lymphoid cells, but mannose 6-phosphate was identified only in enzyme of control cells. Extension of this investigation to lymphoid cultures of a pseudo-Hurler polydystrophy patient also identified only phosphoserine in alpha-L-fucosidase. Using [3H]mannose instead of 32Pi, the precise identification of mannose 6-phosphate in alpha-L-fucosidase of control cells, and its absence in alpha-L-fucosidase of I-cell and pseudo-Hurler cells, was established. The stoichiometry of phosphorylation of alpha-L-fucosidase in I-cell, pseudo-Hurler and control lymphoid cells was 3, 4 and 10 mol Pi/mol enzyme, respectively. alpha-L-Fucosidase was located in lysosomes isolated from control, I-cell and pseudo-Hurler lymphoid cells by subcellular fractionation on Percoll density gradients. Both I-cell and pseudo-Hurler lymphoid cells displayed normal intralysosomal activity of alpha-L-fucosidase despite lack of the mannose 6-phosphate marker. Thus, I-cell and pseudo-Hurler lymphoid cells must possess a mannose 6-phosphate-independent mechanism for directing alpha-L-fucosidase to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

An established mechanism for directing newly made acid hydrolases to lysosomes involves acquisition of mannose 6-phosphate residues by the carbohydrate portion of acid hydrolases followed by binding to specific membrane-bound transport receptors and delivery to lysosomes. Two distinct phosphomannosyl receptors (CI-MPR and CD-MPR) have been identified. Alternative mechanisms for trafficking acid hydrolases exist. This report examines means for the possible receptor-mediated intracellular transport of alpha-L- fucosidase in lymphoid cells. The binding of alpha-L-fucosidase to intact cells and to total cell membrane preparations, in conjunction with immunoassays of solubilized membrane preparations, revealed the presence of CI-MPR and CD-MPR on human lymphoid and fibroblast cell lines. The mean level of CD-MPR in nine lymphoid cell lines was 7.2-fold greater than CI-MPR. The mean level of CI-MPR in two fibroblast lines was 3.8-fold greater than CD-MPR. The mean content of CI-MPR was 19.5-fold greater in the fibroblasts than in the lymphoid cells. The CD-MPR content of fibroblasts and lymphoid cells was nearly equivalent. Among these cell lines were a fibroblast and a lymphoid line from the same individual. These results indicate that human B-lymphoid cells are deficient in CI-MPR and suggest that modulation of expression of CI-MPR and CD-MPR in lymphoid cells differs from that in fibroblasts, including cell lines with identical genomes. No specific receptor capable of binding alpha-L-fucosidase independent of mannose 6-phosphate was demonstrable, despite published results that support the existence of a mannose 6-phosphate independent trafficking mechanism in lymphoid cells for this enzyme.

N-Acetylglucosamine 1-phosphotransferase is a key enzyme required for synthesis of the mannose 6-phosphate recognition marker that is used by many newly made acid hydrolases for their transport to lysosomes. It has previously been found that lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy have nearly normal intracellular and intralysosomal activities of several lysosomal acid hydrolases, despite a deficiency of N-acetylglucosamine 1-phosphotransferase. These results suggest that lymphoid cells may provide an important system to investigate alternate mechanisms for targeting newly made acid hydrolases to lysosomes. In the present study, the biosynthesis, processing and secretion of alpha-L-fucosidase in I-cell and pseudo-Hurler lymphoid cells was used as a model system to study the existence of such mechanisms. The level of intracellular alpha-L-fucosidase protein in exponentially growing I-cell or pseudo-Hurler lymphoid cultures was statistically indistinguishable from the mean of 19 control cultures. A 1.5 h [35S]methionine pulse experiment showed that alpha-L-fucosidase is initially synthesized by I-cell, pseudo-Hurler and control cultures as an intracellular form (Mr = 58,000). Companion cultures chased with methionine from 2 to 21 h processed the enzyme to an intracellular form (Mr = 60,000) and an extracellular form (Mr = 62,000). All enzyme forms were glycoproteins with polypeptide chains of Mr 52,000. In control cells incubated with radioactive inorganic phosphate (32Pi), less than 1% of the 32Pi incorporated into alpha-L-fucosidase was associated with carbohydrate chains and greater than 99% with polypeptide chains. In I-cell disease lymphoid cells, the 32Pi incorporated into alpha-L-fucosidase was associated solely with polypeptide chains. A qualitative analysis of phosphorylated residues identified phosphoserine in alpha-L-fucosidase from control and I-cell lymphoid cells. Only alpha-L-fucosidase from control cells contained mannose 6-phosphate. These results are consistent with the proposal that I-cell lymphoid cells may use a mannose 6-phosphate-independent mechanism for routing alpha-L-fucosidase. Additional metabolic labelling experiments demonstrated the presence of 32P-labelled alpha-L-fucosidase in both cells and medium of a control lymphoid culture, but only in cells of an I-cell lymphoid culture. In contrast, alpha-L-fucosidase labelled with [35S]methionine was found in cells and medium of control and I-cell lymphoid cultures. Since phosphoserine was only found to occur in intracellular, but not in extracellular alpha-L-fucosidase of the I-cell culture, we speculate that phosphoserine may be involved in intracellular retention of alpha-L-fucosidase in I-cell lymphoid cells.

Fucosidosis is an inherited lysosomal storage disease due to a deficiency of alpha-L-fucosidase activity. Exponentially growing lymphoid cell cultures from a fucosidosis patient (JH) had 16-fold lower extracellular alpha-L-fucosidase protein and 72-fold lower intracellular alpha-L-fucosidase protein with negligible catalytic activity as compared with the mean of 19 control cultures. The percentage of total alpha-L-fucosidase protein released extracellularly by JH cells was 71% as compared with 35% +/- 9% for control cells. During a 1.5 h pulse with 35S-methionine, alpha-L-fucosidase was synthesized by JH cells as an intracellular doublet with Mr of 58,000 and 56,000 and by control cells as an intracellular form with Mr = 58,000. During a subsequent 21 h chase with unlabeled methionine, JH alpha-L-fucosidase was entirely secreted. In contrast, only 25%-30% of control enzyme was secreted with the remainder retained intracellularly. Thus, JH lymphoid cells synthesized a reduced amount of alpha-L-fucosidase that was catalytically inefficient and was hypersecreted. Treatment of JH alpha-L-fucosidase with N-glycanase produced polypeptide chains with Mr of 52,000 and 54,000. Previously, treatment of control alpha-L-fucosidase with N-glycancase produced a single polypeptide chain with Mr of 52,000 (Biochem Genet 1988; 26: 401-20). The doublet polypeptide chains of alpha-L-fucosidase in JH cultures may represent expression of two distinct allelic forms of mutant alpha-L-fucosidase.

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.

N-Methyldeoxynojirimycin, 1-deoxymannojirimycin, and monensin interferred with normal processing of asparagine-linked oligosaccharide chains of alpha-L-fucosidase in lymphoid cells by blocking conversion of high-mannose oligosaccharides of newly made precursor enzyme to complex oligosaccharides of mature intracellular and extracellular forms of enzyme. These compounds did not substantially alter the distribution of newly made alpha-L-fucosidase between intracellular and extracellular compartments. Thus, sorting of newly made alpha-L-fucosidase molecules that are retained intracellularly from molecules that are eventually secreted does not require terminal glycosylation or the trimming of glucose or alpha-D-(1----2)-linked mannose residues from carbohydrate chains. Chloroquine and ammonium chloride had no substantial effect on the structural processing or on the intracellular-extracellular distribution of alpha-L-fucosidase in lymphoid cells. In other cell types, these weak bases caused a massive secretion and an intracellular deficiency of acid hydrolases. The different responses to weak bases in lymphoid cells and the other cell types can be explained either by an inability of these agents to neutralize the pH of intracellular organelles in lymphoid cells or by a routing mechanism in lymphoid cells that is independent of pH.

The quantity of alpha-L-fucosidase activity in human serum is determined by heredity. An individual may inherit either low, intermediate, or high serum enzyme activity. An enzyme-linked immunoabsorbent assay has been developed that can detect 0.3 ng of alpha-L-fucosidase protein. Enzyme protein in serum of 102 individuals ranged from 20 to 835 ng/ml. The group included individuals with low, intermediate, and high enzyme activity. The specific activity of alpha-L-fucosidase within this group was statistically the same (mean +/- SD = 11,002 +/- 1051 U/mg). Thus, individuals with low and intermediate enzyme activity in serum had lower amounts of enzyme protein with the same specific activity as in individuals with high enzyme activity. Fucosidosis is a rare inherited disease in which alpha-L-fucosidase activity in tissues and body fluids is low or absent. The concentrations of enzyme protein in sera of a fucosidosis patient and parents were 76,565, and 604 ng/ml, respectively, and the specific activities of enzyme were 1316, 8938, and 8858 U/mg, respectively. Thus, the fucosidosis serum probably contained a structurally altered enzyme with reduced catalytic activity. The somewhat low specific activities in the parents suggested that their sera contained both structurally altered and normal protein.

In humans, the quantity of alpha-L-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high alpha-L-fucosidase in serum were established. Steady-state levels of intracellular and extracellular alpha-L-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus, in vivo serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made alpha-L-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with 35S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with a Mr = 58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form of Mr = 60,000 and an extracellular form of Mr = 62,000. All three enzyme forms were glycoproteins with a common polypeptide chain of Mr = 52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of alpha-L-fucosidase was found. Fucosidosis is a rare, inherited disease in which alpha-L-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular alpha-L-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (Mr = 52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme.

Sera from a group of patients with ovarian cancer had a statistically significant deficiency of alpha-L-fucosidase activity compared with sera from healthy females or female patients with cervical or breast cancer. Mixing experiments did not identify an inhibitor of alpha-L-fucosidase activity in the sera of ovarian cancer patients. Decreased activity of alpha-L-fucosidases was not associated with stage of disease, tumor burden, histologic type, or grade of differentiation. Unlike alpha-L-fucosidase, beta-man-nosidase and beta-N-acetylglucosamindase in sera of ovarian cancer patients were not deficient in activity. Examination of population data of healthy females and of pedigrees of ovarian cancer patients suggested that the quantitative activity alpha-L-fucosidase in serum was genetically determined. Of 60 healthy females, 4 had low enzyme activity (less than 100 U alpha-L-fucosidase/ml serum), 26 had intermediate activity (100-274 U alpha-L-fucosidase/ml), and 30 had high activity (275 U/ml), whereas of 44 ovarian cancer patients, 11 had low, 29 had intermediate, and 4 had high activity. Application of the Hardy-Weinberg law to these data revealed that low enzyme activity in sera was three times more prevalent in the ovarian cancer group, the allele for this low enzyme activity being two times more common. These observations suggested that deficiency of alpha-L-fucosidase activity in sera of females may be a hereditary condition associated with increased risk for development of ovarian cancer.

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of alpha-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular alpha-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular alpha-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total alpha-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35 +/- 9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains of Mr = 52,000. During a 1.5-hr pulse-label with 35S-methionine, alpha-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form with Mr = 58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form with Mr = 62,000. In contrast, only 25-30% of control enzyme was processed to an extracellular form (Mr = 62,000), with the remainder retained intracellularly (Mr = 60,000). In the other two fucosidosis cell lines, alpha-L-fucosidase was synthesized as an intracellular form with Mr = 56,000 that was processed to an extracellular form with Mr = 60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of alpha-L-fucosidase as expressed by lymphoid cells.

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.

alpha-L-Fucosidase from serum of humans with either high or low enzyme activity was separately purified. the enzyme from either source had virtually the same heat stability and pH activity profile. It has been widely reported that alpha-L-fucosidase in crude sera from individuals with high and low enzyme activity differed with respect to heat stability and activity at pH 4 relative to activity at pH 5, the pH optimum of the enzyme. We investigated this discrepancy and found that both the heat stability and relative activity at pH 4 of alpha-L-fucosidase from sera with either high or low enzyme activity was dependent upon enzyme concentration. With decreasing enzyme concentration, the enzyme was more heat labile and had less relative activity at pH 4. Consequently, if the data obtained using high and low enzyme activity sera are compared on the basis of equivalent amounts of serum instead of equivalent amounts of enzyme activity, differences between the enzyme from high and low activity serum would be erroneously inferred. Apparently, this is what other investigators have done. Moreover, we found that alpha-L-fucosidase can exist in heat-stable or labile species with sedimentation coefficients of 9.8 S and 4.8 S, respectively. The interconversion and relative proportion of these species is dependent upon enzyme concentration and pH.