Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo.

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed "recoding". In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for -1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed -1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.

Bacteroides fragilis fucosidases facilitate growth and invasion of Campylobacter jejuni in the presence of mucins.

The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilisation. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion.

Alpha-L-fucosidase-1 is a diagnostic marker that distinguishes mucoepidermoid carcinoma from squamous cell carcinoma.

Alpha-L-fucose is a component of glycans on the cell surface. Alpha-L-fucose is correlated with tumorigenesis and malignancy, and alpha-L-fucosidase-1 (FUCA1), the enzyme that removes terminal α-L-fucose residues from glycoproteins, is downregulated in some high malignancy cancers. The expression profile of FUCA1 in head and neck tumors remains unknown, and we analyzed the expression profiles of FUCA1 and an upstream molecule p53 in mucoepidermoid carcinoma (MEC) and oral squamous cell carcinoma (OSCC). FUCA1 was expressed in most MECs irrespective of histopathological grading, whereas expression in OSCCs was low. High immunohistochemical intensity of p53 was detected in OSCCs at high frequency, but rarely detected in MECs. Genetic mutation analysis using next-generation sequencing revealed no significant mutation of TP53 in MECs. We further analyzed the expression profiles of FUCA1 in normal major and minor salivary glands and found strong expression in the intercalated duct, moderate expression in mucous acinar cells and no expression in serous acinar cells. These contrasting immunohistochemical profiles and anatomical distribution in normal salivary glands suggest that FUCA1 is a useful marker to distinguish MEC from OSCC, and many MECs have similar immunohistochemical phenotypes to intercalated duct and mucous acinar cells.

Purification, expression and characterization of a novel α-l-fucosidase from a marine bacteria Wenyingzhuangia fucanilytica.

α-l-Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α-l-Fucosidases, glycoside hydrolases for catalyzing the removal of α-l-fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α-l-fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α-l-fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α-l-fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α-l-fucosidases.

Novel p53 target gene FUCA1 encodes a fucosidase and regulates growth and survival of cancer cells.

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α-l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.

Cloning, characterization, and production of three α-L-fucosidases from Clostridium perfringens ATCC 13124.

α-L-Fucosidases are key enzymes for the degradation of intestinal glycans by gut microbes. In this work, three putative α-L-fucosidases (Afc1, Afc2, and Afc3) genes from Clostridium perfringens ATCC 13124 were cloned and expressed in Escherichia coli. Afc1 had the α-L-fucosidase domain of glycoside hydrolase (GH) 29 family but showed no enzyme activity toward all the substrates examined. The putative acid/base residue of Afc1, Ser205, was replaced by a glutamic acid which is conserved in GH29-B α-L-fucosidases. However, the mutant Afc1-S205E still failed to show enzyme activity. Afc2 and Afc3 were determined to be 1,3-1,4-α-L-fucosidase of GH29-B subfamily and 1,2-α-L-fucosidase of GH95 family, respectively, and both of them could release fucose from porcine gastric mucin (PGM). When C. perfringens ATCC 13124 grew with the presence of PGM, the transcription of afc1 decreased slightly, while those of afc2 and afc3 increased to 2.2-fold and 1.4-fold, respectively, and the enzyme activities of Afc2 and Afc3 in the culture increased to 2.2-fold and 2.6-fold, respectively. These results suggest that Afc2 and Afc3 are involved in the degradation of intestinal fucosyl glycans by C. perfringens ATCC 13124.

Determination of lysosomal exoglycosidases in human saliva.

BACKGROUND: Currently we observe a growing interest in human saliva as a non-invasive material for diagnosis and monitoring of general and oral diseases. METHODS: The aim of our study was adaptation of the Marciniak et al. (Marciniak J, Zalewska A, Popko J, Zwierz K, 2006, Clin Chem Lab Med 44: 933-937) method for determination of HEX and GLU activity in synovial fluid, and for determination of: HEX and GLU, as well as MAN, GAL, and FUC activity in human saliva. RESULTS: Under optimal conditions, 10 µl of saliva for HEX, and 30 µl for GLU, MAN, GAL and FUC, were sufficient for determination of human salivary exoglycosidases activity with variation coefficient ranging from 0.89 for GLU to 0.99 for GAL. CONCLUSION: The adapted method for exoglycosidases activity determination in human saliva is sufficiently sensitive and precise to use in clinical diagnosis.

Recombinant expression of Drosophila melanogaster α-L-fucosidase in Trichoplusia ni cells.

A cDNA encoding an α-l-fucosidase from Drosophila melanogaster was obtained from the recombinant plasmid named pGEM-DmFuca and inserted into the pBacHTeGFPT vector to construct the recombinant donor plasmid which was transposed to the target AcBacmid in Escherichia coli (DH10) by Tn7 transposition function. The AcBacmid-GFP-DmFuca plasmid was used to transfect Tn-5B1-4 cells of the Cabbage looper Trichoplusia ni. SDS-PAGE analysis revealed a band of about 80kDa. Using a polyclonal antiserum raised against α-l-fucosidase protein from D. melanogaster Western blotting analysis confirmed that the fusion protein eGFP-DmFuca has been successfully expressed in a biologically active form in Tn-5B1-4 cells. The recombinant protein, containing the histidine-tag motif, was purified using an affinity chromatography column. In vitro binding assays the purified eGFP-DmFuca interacts with α-l-fucose residues present on the micropyle of the D. melanogaster eggshell, confirming that the α-l-fucosidase is a good candidate as receptor involved in gamete interactions in fruit fly.

1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism.

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.

The alpha-L-fucosidase from Sulfolobus solfataricus.

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups alpha-L-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first alpha-L-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed -1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for beta and alpha glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus.

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.

Combined use of established and novel tumour markers in the diagnosis of head and neck squamous cell carcinoma.

Serum SCC, CYFRA 21-1, and CEA are the common tumour markers for head and neck squamous cell carcinoma (HNSCC), although diagnostic sensitivity should be yet improved, especially at early stages. In the present study, we have reported the diagnostic value of two novel serum tumour markers in HNSCC: alpha-L-fucosidase (AFU) activity, and total sialic acid concentration adjusted by total protein concentration (TSA/TP). Using the cut-off 4.0 U/ml, AFU showed a sensitivity of 55% with specificity levels of 91%, 85% and 50% to discriminate HNSCC patients from healthy donors, drinking and smoking subjects, and patients with benign diseases, respectively. Furthermore, AFU showed the best sensitivity (71%) in the detection of patients with premalign lesions. Using the cut-off 12.0 ng/mg, TSA/TP showed the best sensitivity levels (63%) in the diagnosis of HNSCC with specificity levels of 94%, 50% and 90%, regarding healthy donors, drinking and smoking subjects, and patients with benign diseases, respectively. It was of special interest that sensitivity in the diagnosis of HNSCC at non-disseminated stages was improved when using combinations of AFU+CYFRA or TSA/TP+CYFRA, up to 86% or 71% in TNM I, 60% or 80% in TNM II, and 80% or 60% in TNM III, respectively.

[Change of isoforms' spectrum of alpha-L-fucoside secreted by affected cells in some hereditary lysosomal diseases]. / Izmenenie spektra mnozhestvennykh form alpha-fukozidazy, sekretiruemoí kletkami-misheniami pri nekotorykh nasledstvennykh énzimopatiiakh.

In vitro it was studied the isoform spectra of the intracellular and secreted alpha-L-fucosidase from skin fibroblasts of patients with Fabry disease (glycolipidosis), Hurler and Sanfilippo D diseases (mucopolysaccharodosis, types I and III) and in the normal state was studied. It was shown that the multiple form profile of secreted alpha-L-fucosidase in patients fibroblasts was changed as compared to that in control: the pathological cells were characterized by expression of more basic isoforms of alpha-L-fucosidase. The changes were similar to those in sucrose-loaded normal cells, modelling storage disease. The data obtained allow the suggestion that the intracellular accumulation of compounds whose hydrolysis was disturbed on a hereditary deficiency of enumerated glycosidases can influence the posttranslational processing of alpha-L-fucosidase, the enzyme which is not primary affected in these disorders. These data allow the conclusion that the high phenotypic heterogenity of lysosomic storage diseases is possibly due to the influence of so-called epigenetic factors involving the changes in properties of such glycosidases as are not associated with a primary hereditary defect.

Glycoside hydrolase production by an anaerobic rumen fungus Caecomyces communis.

The ruminal fungus Caecomyces communis was grown anaerobically either in a discontinuous cultivation system or in a fermentor with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The best culture conditions for glycoside hydrolase production were obtained in Lowe medium with daily fresh medium addition, whereas the Orpin medium with ruminal fluid was favourable to fungal growth and to the enzyme export process. Among glycoside hydrolases assessed in both culture fluid and cellular homogenate, beta-D-fucosidase activity was preponderant. Most studied enzymes were mainly associated with cells (from 50% to 99%). Glycoside hydrolase activities were constitutive, but their level was regulated by a carbon source. beta-D-fucosidase and beta-D-xylosidase activity production was activated by the association of glucose plus cellobiose, whereas beta-D-glucosidase activity production was stimulated by cellobiose alone. Enzyme release could be favoured by glucose alone or by Ray grass hay added to glucose plus cellobiose.

The production of neuraminidase and fucosidase by Helicobacter pylori: their possible relationship to pathogenicity.

The pathogenicity of enterobacteria often correlates with their production of neuraminidase (sialidase). Forty-nine Helicobacter pylori isolates have therefore been examined for their production of neuraminidase and other glycosidases. All 49 isolates produced considerable neuraminidase (median 228 IU/microg protein, interquartile range 121-370), pH optimum 7.5. Nine of the 49 also produced fucosidase (median 23 IU/microg protein, interquartile range 12-39), pH optimum 7.0. Production of these enzymes did not correlate with bacterial Cag A expression or duodenal ulceration. Neutrophils exposed to neuraminidase show increased adherence to endothelium so the neuraminidase production by H. pylori could partly explain the predominant neutrophil inflammatory infiltrate seen in H. pylori-associated gastritis. Inhibition of this enzyme by use of neuraminidase-inhibitors could be a useful therapeutic approach.

Efficient production and purification of extracellular 1,2-alpha-L-fucosidase of Bacillus sp. K40T.

When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-alpha-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5-7.0 and was stable at pH 6.0-9.0. The enzyme hydrolyzed the alpha-(1-->2)- L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I 2)-O-beta-D-galactose-(1-->3)-N-acetyl-O-beta-D- glucosamine-(1-->3)-O-beta-D-galactose-(1-->4)-D-glucose>, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze alpha-(1-->3)-, alpha-(1-->4)- and alpha-(1-->6)-L-fucosidic linkages in LNF-III 4)[O- alpha-L-fucose-(1-->3)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose>, LNF-II 3)[O-alpha-L- fucose-(1-->4)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose> or 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine.

Synthesis and processing of rat sperm-associated alpha-L-fucosidase.

alpha-L-Fucosidase (alpha-fucosidase) is the most active of a number of glycosidases measured in rat epididymal sperm through use of artificial 4-methylumbelliferyl substrates. In addition, enzyme activity can be detected in purified populations of testicular germ cells; in comparison to round spermatids, caput epididymal sperm show at least a 1.8-fold increase in alpha-fucosidase activity per cell. Metabolic labeling of cultured testicular germ cells, followed by immunoprecipitation and SDS-PAGE, reveals synthesis of alpha-fucosidase as a polypeptide of 54 kDa in pachytene spermatocytes and round spermatids but no synthesis in condensing spermatids. This polypeptide is N-glycosylated but does not undergo further processing as determined by pulse/chase labeling and treatment with endoglycosidase H. In contrast, the alpha-fucosidase synthesized by cultured clone 9 cells (a rat liver-derived cell line) undergoes processing from a 54-kDa precursor to a slightly larger intermediate centered at 56 kDa on SDS-PAGE (via carbohydrate modification) and finally to a 52-kDa mature polypeptide. Immunoblotting confirms the presence of the 54-kDa form of alpha-fucosidase in testicular germ cells and shows the existence of a 52-kDa mature polypeptide in epididymal sperm. In addition, immunoreactive polypeptide is more prominent in caput epididymal sperm preparations; this is consistent with the increase in enzyme activity. In the absence of alpha-fucosidase synthesis beyond the round spermatid stage of development, it appears that alpha-fucosidase may be acquired from the luminal fluid by sperm during transit through the excurrent duct system. This hypothesis is supported by evidence that metabolically labeled cultured epididymal epithelial cells synthesize and secrete into culture medium an immunoprecipitable 58-kDa alpha-fucosidase polypeptide. Analysis of sperm isolated from the first part of the caput epididymis indicates that both acquisition and processing of sperm-associated alpha-fucosidase takes place prior to or concomitant with arrival of sperm in the epididymis.

Biosynthesis, processing, and secretion of alpha-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy.

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.

Hereditary ovarian cancer. Pedigree studies, Part II.

Hereditary ovarian carcinoma is heterogenous. There are at least three genetic variants, namely, hereditary site-specific ovarian carcinoma, hereditary breast/ovarian carcinoma syndrome, and Lynch syndrome II. Early age of onset characterizes these disorders. A crucial hallmark of these disorders is the integral association of extraovarian cancers, such as carcinoma of the endometrium and colon in Lynch syndrome II. We have described 24 pedigrees of ovarian cancer-prone families in order to depict the several differing heterogenous variants. Interest in hereditary ovarian cancer has increased remarkably, due in part to the fact that its surveillance has been wholly unsatisfactory, as have therapeutic measures. Prevention through prophylactic oophorectomy offers hope. However, there is a risk for extraovarian peritoneal serous papillary carcinoma, consonant with primary cancer of the ovary. This must be discussed with these at-risk patients. Until a biomarker of acceptable sensitivity and specificity is identified, the family history must remain the key to hereditary ovarian cancer diagnosis.

Synthesis and processing of lysosomal alpha-fucosidase in cultured human fibroblasts.

The lysosomal enzyme alpha-L-fucosidase from human skin fibroblasts is synthesized as a 53 kDa glycosylated precursor which is then proteolytically processed to a 50 kDa mature form. This was confirmed by pulse-chase labeling studies with chase times up to 72 h. In fibroblasts treated with 1-deoxymannojirimycin to prevent trimming of high mannose oligosaccharides, endoglycosidase H (endo H) treatment completely deglycosylated and reduced the size of immunoprecipitated alpha-fucosidase by 4-5 kDa, suggesting the presence of two oligosaccharide units. Endoglycosidase H and endo F studies on untreated alpha-fucosidase suggested the presence of one complex-type and one high mannose-type unit, and that the final processing from 53 to 50 kDa did not involve the removal of carbohydrate. Processing was inhibited by the thiol proteinase inhibitor Ep-459, but not by Ep-475 or leupeptin. Since Ep-459 treatment increased both alpha-fucosidase activity (3-fold) and the amount of immunoprecipitable alpha-fucosidase protein in normal human skin fibroblasts, this suggests a role for cysteine-like proteinases either directly or indirectly in lysosomal hydrolase processing and turnover. Subcellular fractionation studies revealed that the proteolytic processing of the 53 kDa precursor to the 50 kDa mature form occurred in the lysosome, or some other dense organelle.