Analysis of cathepsin D in human breast cancer: usefulness of the processed 31 kDa active form of the enzyme as a prognostic indicator in node-negative and node-positive patients.

The relative amounts of the precursor (52 kDa) and processed (31,27 kDa) forms of cathepsin D have been analyzed by Western blotting in biopsied breast tissue cytosols from 134 lesions from invasive breast cancer patients, 24 lesions from patients with ductal carcinoma in situ (DCIS), 227 lesions from benign breast disease patients, and 28 lesions from normal control subjects. The mean relative percentage amount of the 31 kDa form was significantly increased (p < 0.001) in the invasive breast cancer group compared to the other three groups. In addition, the mean relative percentage amount of the 31 kDa form was significantly increased (p < 0.05) in node-positive compared to node-negative breast cancer patients. In the benign breast disease group, patients with proliferative-type disease had a significantly increased (p = 0.02) mean relative percentage amount of the 31 kDa form of cathepsin D compared to patients with nonproliferative-type disease. Invasive breast cancer patients were followed for up to 75 months to determine if the relative percentage amount of the 31 kDa form of cathepsin D was predictive of disease-free and overall survival. Although the amount of the 31 kDa form was not predictive of disease-free survival, patients in the 'high' 31 kDa group (> 18%) were significantly (p < 0.05) more likely to die than patients in the 'low' 31 kDa group (< or = 18%). The 12 patients who died were all node-positive and in the high 31 kDa group. It thus appears that the relative amount of the processed, active 31 kDa form of cathepsin D is a useful prognostic indicator, at least in node-positive breast cancer patients.

Characterization of human semen alpha-L-fucosidases.

Human semen contains a large amount of alpha-L-fucosidase activity, the great majority of which is found in the seminal fluid. Immunocytochemical studies indicate that a small amount of semen fucosidase activity is present on the sperm plasma membrane, primarily in the posterior head region. Subcellular fractionation studies also indicate that sperm alpha-L-fucosidase is present in the plasma membrane-enriched fraction. Comparative characterization of human seminal fluid and sperm alpha-L-fucosidases indicates that seminal fluid alpha-L-fucosidase has a broad pH optimum curve with a number of near-equal maxima between pH 4.8 and 7.0 while sperm fucosidase has a major optimum between pH 3.4 and 4.0. Isoelectric focusing indicates that seminal fluid alpha-L-fucosidase contains three to six isoforms with isoelectric points (pI) of 5-7 while sperm fucosidase contains two distinct isoforms with pI values of 5. 2 +/- 0.2 and 7.0 +/- 0.2. Western blotting indicates that seminal fluid fucosidase contains a major protein band with a molecular mass ratio (M(r)) of approximately 56 kDa while sperm fucosidase contains a major protein band of approximately 51 kDa. The overall results indicate the presence of a low-abundance, plasma membrane-associated human sperm alpha-L-fucosidase, which is different in its properties from human seminal fluid alpha-L-fucosidase(s), and whose function is not yet known.

Characterization of purified cathepsin D from malignant human breast tissue.

The aspartyl protease cathepsin D (EC 3.4.23.5) appears to be found in increased amounts and/or abnormally secreted in breast cancer cells, and may contribute to the metastatic spread of malignancy. In the present study, cathepsin D was purified 4800-fold in 20% yield from malignant human breast tissue using affinity chromatography on pepstatin-agarose and DEAE-Sephadex chromatography. Slab gel SDS-PAGE of the purified cathepsin D indicated the presence of three major protein bands (31, 13, 12, kDa) and two minor protein bands (47, 29 kDa). Western blotting indicated that the 31 kDa band was the major immunoreactive species. Isoelectric focusing indicated that the purified cathepsin D consisted of three major isoforms at approximate pIs of 7.4, 7.0 and 6.6, and a possible isoform of lower activity centered around pI 3.2. The pH curve of purified cathepsin D indicated a broad optimum centered around pH 3.4. Lectin blotting suggested the presence of mannose residues but no evidence was found for lectin-available sialic acid, fucose, N-acetylglucosamine and galactose residues. The investigated properties of purified cathepsin D from malignant breast tissue are very similar, if not identical, to the properties of cathepsin D previously purified from normal human breast tissue. Our findings suggest that the elevated activity and antigenic levels of cathepsin D in malignant breast tissue are due to increased amounts of apparently normal enzyme.

Alteration of the isoform composition of plasma-membrane-associated rat sperm alpha-L-fucosidase during late epididymal maturation: comparative characterization of the acidic and neutral isoforms.

In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum alpha-L-fucosidase in rat sperm. In the present study, rat sperm alpha-L-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of alpha-L-fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53+/-2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of alpha-L-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. alpha-L-Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and alpha-L-fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5.2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sperm from the distal cauda were separated by isoelectric focusing and comparatively characterized. They had similar pH-activity curves (with optima near pH 7) and comparable apparent KM values (0.4+/-0.04 mM) for 4-methylumbelliferyl alpha-l-fucopyranoside. Preincubation of the isoforms at different temperatures indicated that isoform A is considerably more thermostable than isoform B. Immunoprecipitation studies using polyclonal antibodies against human liver alpha-L-fucosidase indicated that approx. 90% of the enzymic activity for both isoforms was immunoprecipitable under conditions that immunoprecipitated essentially all the human liver enzyme. Neuraminidase treatment of sperm alpha-L-fucosidase from distal cauda (when compared with the appropriate heat-treated control) led to disappearance of isoform A and a concomitant increase in isoform B. The overall results suggest that isoform A is derived by sialylation of isoform B near the end of epididymal maturation.

Purification and characterization of cathepsin D from normal human breast tissue.

The lysosomal aspartyl protease cathepsin D is present in most mammalian cells and is active in the catabolism of intracellular and endocytosed proteins. It appears to be overexpressed and abnormally secreted in breast cancer cells, and may contribute to the process of tumor metastasis. In the present study, cathepsin D was purified 4500-fold from normal human breast tissue using pepstatin-agarose, DEAE Sephadex, and Sephadex G-75 chromatography. The resulting enzyme on SDS-PAGE contained five protein bands (47, 31, 29, 13, and 12kDa) which were all immunoreactive on western blot analysis using anti-cathepsin D polyclonal antibodies. The isoform profile of purified cathepsin D consisted of three major peaks at approximate pI 7.3, 6.8, and 6.3, and a broad area of lower activity between pI of 5.0 and 2.0. The purified enzyme had a broad pH optimum centered around pH 3.3. Lectin blotting indicated that cathepsin D is a glycoprotein which is recognized by Galanthus nivalis agglutinin and concanavalin A, suggesting the presence of mannose residues. However, Sambucus nigra agglutinin, Tetragonolobus purpureas agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lectin-available sialic acid, fucose, N-acetylglucosamine, and galactose residues, respectively.

Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum alpha-L-fucosidase from rat testis and epididymal spermatozoa.

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum alpha-L-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for alpha-L-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the alpha-L-fucosidase activity was associated with the 48,000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of alpha-L-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH-activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm-egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm-egg interactions.

Western blotting and isoform analysis of cathepsin D from normal and malignant human breast cell lines.

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and isoform composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of cathepsin D for all three cell lines occurs at 31 kDa. The cancer cell lines contain significant amounts of cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such pIs. In addition, the cancer cell lines contain two to three major isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analysis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered acid protease isoform profiles with increased relative amounts of pepstatin-inhibitable and immunoreactive acid protease activity (cathepsin D) compared to normal breast tissue or cells.

Peptide mapping of the subunits and deglycosylated polypeptides of human liver alpha-L-fucosidase.

The subunits of human liver alpha-L-fucosidase have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, excised, and subjected to peptide mapping after CNBr cleavage or trypsin digestion. The CNBr peptide maps of the glycosylated 56- and 51-kDa subunits were similar except that the larger subunit had several peptides with M(r)s shifted higher than those apparent for the smaller subunit. These M(r) differences were almost completely eliminated when CNBr peptide mapping was performed on the deglycosylated 48- and 45-kDa polypeptides, suggesting that the M(r) differences were due to carbohydrate differences. Minor differences not related to glycosylation were found in the CNBr peptide maps for the 48- and 45-kDa polypeptides including the presence of small amounts of three peptides in the larger polypeptide not found in the smaller polypeptide. Sequence analysis suggested that both the 48- and the 45-kDa polypeptides were blocked at their amino-termini but analysis of the largest CNBr peptide from each polypeptide indicated an identical 13-amino-acid sequence corresponding to residues 6 through 18 from the cDNA-deduced sequence of mature alpha-L-fucosidase. Tryptic peptide mapping indicated very similar HPLC peptide profiles for the deglycosylated 48- and 45-kDa polypeptides except for the presence of small amounts of six peaks present in the larger polypeptide which were not detected in the smaller polypeptide. The overall results provide the first evidence that the polypeptides of human liver fucosidase are very similar and probably encoded by the same gene. However, minor differences in the polypeptides exist, possibly due to normal allelic variation, alternative splicing, proteolytic processing, and/or posttranslational modifications other than those due to glycosylation.

Structural characterization of the N-glycans of a humanized anti-CD18 murine immunoglobulin G.

This study characterized the N-glycans of a humanized immunoglobulin G4 (IgG4) expressed in NS/O mouse myeloma cells and directed against the CD18 family of adhesion-promoting receptors on leukocytes. The N-glycans were released from the purified recombinant IgG by N-glycanase treatment, purified by Sephadex G50 chromatography, and fractionated by Bio-Gel P-4 chromatography into three oligosaccharide pools. Each pool was analyzed individually by glycosyl composition analysis, high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), 600-MHz 1H-NMR spectroscopy, and electrospray-ionization mass spectrometry. In addition, each of the three pools was subfractionated by HPAEC and the isolated subfractions that contained sufficient material were hydrolyzed and analyzed for glycosyl composition by HPAEC-PAD. The overall results indicate the presence of five oligomannoside-type structures (containing 5 to 8 Man residues) which are not usually found in IgG, and the presence of eight diantennary (mostly truncated) N-acetyllactosamine-type structures which are typical of mouse and human IgGs. The N-acetyllactosamine-type structures were heterogeneous with regard to alpha(1-->6) fucosylation of the linkage GlcNAc, and the presence or absence of GlcNAc and/or Gal beta(1-->4)GlcNAc extending the core pentasaccharide (Man3GlcNAc2). No evidence was found for the presence of sialic acid or bisecting GlcNAc residues on the N-acetyllactosamine-type chains. The latter finding suggests that the N-glycans of this humanized IgG are of the mouse type.

Western blotting and enzymatic activity analysis of cathepsin D in breast tissue and sera of patients with breast cancer and benign breast disease and of normal controls.

Increased total antigen amounts of cathepsin D in breast tissue have been reported to be associated with increased disease recurrence, more frequent metastasis, and increased mortality in breast cancer patients. In the present study, Western blotting analysis has been used for the first time to determine the relative amounts of precursor and processed forms of cathepsin D in sera and breast tissue of patients with breast cancer, benign breast disease, and normal controls. Sera gave similar blots for breast cancer patients and controls with two major forms of cathepsin D (M(r) 52,000 and 27,000). Malignant breast tissue contained the two forms of cathepsin D found in sera and an additional M(r) 31,000 form which was found in significantly increased (P < 0.001) relative amounts in breast tissue from 43 breast cancer patients [24 +/- 12% (SD)] when compared to 51 benign breast disease patients (13 +/- 8.9%) and 23 normal controls (1.8 +/- 4.4%). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.41) in relative amounts of the M(r) 31,000 form of cathepsin D between proliferative-type and non-proliferative-type fibrocystic breast disease. A cathepsin D assay has been optimized for human breast tissue and used to demonstrate for the first time significantly increased (P < 0.001) amounts of pepstatin-inhibitable, cathepsin D-specific activity in breast tissue from 36 breast cancer patients (2.2 +/- 1.4 units/mg of protein) when compared to 47 benign breast disease patients (0.63 +/- 0.43) and 23 normal controls (0.24 +/- 0.21). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.21) in pepstatin-inhibitable, cathepsin D-specific activity between proliferative-type and nonproliferative-type fibrocystic breast disease. The positive correlation (r = 0.82) of increased amounts of the M(r) 31,000 form of cathepsin D and increased pepstatin-inhibitable, cathepsin D enzymatic activity in malignant breast tissue suggests that the M(r) 31,000 form is the proteolytically active form of the enzyme which may be involved in the development and/or metastatic spread of breast cancer.

Immobilized pH gradient focusing of alkaline proteins: analysis of the isoform composition of purified human non-secretory ribonucleases from kidney, liver and spleen.

Previous studies on the isoform composition of human ribonucleases (RNAases) have resulted in confusing and inconsistent results, presumably due to methodological problems in electrofocusing of alkaline proteins. In the present study, immobilized pH gradient (IPG) carrier ampholyte (CA) isoelectric focusing (IEF) and conventional CA-IEF have been evaluated for the analysis of the isoforms of human non-secretory RNAases purified from kidney, liver and spleen. CA-IEF proved unsuitable since the alkaline RNAase isoforms migrated into the cathode. IPG-CA-IEF, however, resolved the RNAase isoforms and marker proteins in the basic region of the gel matrix. The three RNAases had comparable isoform profiles, each with two protein bands with approximate pI values of 10.3 and 10.4. Western blotting showed that the two protein bands of each RNAase were immunoreactive (with polyclonal antibodies that recognize RNAase), indicating that the protein bands are RNAase isoforms. The present results provide reliable pI data on human RNAase isoforms and suggest that IPG-CA-IEF should be a suitable technique for analysing the isoforms of other alkaline proteins.

Rodent tissue alpha-L-fucosidases: analysis of brain and spleen isoforms and characterization of the purified hamster liver enzyme.

1. Isoelectric focusing of brain and spleen alpha-L-fucosidases from four rodents (rat, mouse, guinea-pig, hamster) indicated that only mouse and hamster tissues contained isoforms with significant amounts of activity above pI 7.0. 2. Hamster liver alpha-L-fucosidase was purified approximately 57,000-fold in 80% yield (to a final specific activity of 24,700 nmol/min/mg protein) by affinity chromatography on agarose-epsilon-aminocaproyl-fucosamine. 3. SDS-PAGE analysis of hamster liver alpha-L-fucosidase indicated the presence of one to two closely-spaced subunits at 56 and 60 kDa. Western blotting analysis indicated the hamster enzyme was recognized by polyclonal antibodies but not by a monoclonal antibody (both antibodies prepared against human liver alpha-L-fucosidase). 4. The pH-activity curve of hamster liver alpha-L-fucosidase is broad with an optimum centered around pH 6.8 and with high activity between pH 5.5 and 7.5.

Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes.

Human non-secretory neutral ribonucleases (RNases) from kidney, liver and spleen have been purified and characterized. SDS-PAGE indicates that all three RNases are highly purified and have apparent mol. wts of 17-18 kDa. Kinetic analysis indicates that all three RNases have a broad pH optimum centred around 6.5, and all three have similar substrate specificities with significant preference for RNA and poly(U) when compared to poly(C), poly(A) and poly(G). All of the above data, as well as immunoblotting data using three polyclonal antibodies (anti-human liver RNase, anti-human pancreatic RNase, anti-human eosinophil-derived neurotoxin), indicate that the three proteins are highly purified and are non-secretory RNases (IIN). Further characterization by cyanogen bromide peptide mapping and extensive lectin blotting indicated no significant differences between the three human RNases. All three RNases appear to have very similar, if not identical, protein backbones and all three are glycoproteins which are recognized by lectins with specificity for GlcNAc, Fuc and, to a lesser extent, with specificity for Gal beta(1-4)GlcNAc. No significant tissue-specific differences were found among the three human non-secretory RNases.

Human non-secretory ribonucleases. II. Structural characterization of the N-glycans of the kidney, liver and spleen enzymes by NMR spectroscopy and electrospray mass spectrometry.

The N-glycans have been removed by peptide-N-glycosidase F (PNGase F) from purified human non-secretory RNases derived from kidney, liver and spleen. The spleen RNase was purified by two procedures, one of which did not include the usual acid treatment step (0.25 M H2SO4, 45 min, 4 degrees C), to determine if acid treatment alters the carbohydrate moieties. The N-glycans of the RNases were fractionated by Bio-Gel P-4 chromatography and analysed by 600 MHz 1H-NMR spectroscopy and electrospray mass spectrometry. All four non-secretory RNase preparations contained the following structures: [formula: see text] The relative amounts of the trisaccharide, pentasaccharide and hexasaccharide appeared to vary slightly in the different tissue RNases. The overall results indicate: (i) that acid treatment during purification does not alter the N-glycans of non-secretory RNases; (ii) that the N-glycans from kidney, liver and spleen non-secretory RNases are very similar, if not identical, to one another, but different from the N-glycan structures reported for secretory RNase.

Analysis of the subunits, isoforms and substrate specificity of mouse liver alpha-L-fucosidase.

1. SDS-PAGE indicates the presence of two major protein bands (57 and 62 kDa) for mouse fucosidase and Western blotting indicates that both bands are immunoreactive with polyclonal antibodies (PAbs) and/or monoclonal antibodies (MAbs) raised against human liver fucosidase. The lectins SNA and GNA recognized both mouse protein bands, indicating that both subunits are glycosylated and contain sialic acid residues. 2. Polyacrylamide gel-isoelectric focusing (PAG-IEF) indicated that mouse liver fucosidase contains at least seven isoforms, with three isoforms above pI 6.0, which were not detected in human liver fucosidase. Blotting indicates that the PAbs recognized seven mouse fucosidase isoforms (pIs 3.6-6.8) whereas the four MAbs did not appear to recognize any of the mouse isoforms. 3. The subunit composition of the separated isoforms of mouse alpha-L-fucosidase was investigated by SDS-PAGE. One-to-two closely-spaced protein bands are found in each isoform with a trend of increasing relative amounts of the high-M(r) band in the more acidic isoforms relative to the more neutral isoforms. 4. Human and mouse liver alpha-L-fucosidases hydrolyze L-Fuc from oligosaccharides and glycolipids at comparable rates, with the exception of ganglioside Fuc-GMI which was hydrolyzed by human, but not by mouse, alpha-L-fucosidase.

Differential isoform profiles of alpha 2-macroglobulin from plasma of patients with chronic-progressive or relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is a human neurological disease for which no clinically useful marker has been identified in blood. This study examined alpha 2-macroglobulin (alpha 2M) from the plasma of six patients with chronic-progressive MS and six with relapsing-remitting disease. The alpha 2M trypsin-binding activity in the plasma from both groups of patients did not differ from normal controls. However, after column isoelectric focusing, consistently less alpha 2M activity was recovered from the MS samples: those from the chronic-progressive and relapsing-remitting disease groups were an average of 43% and 68%, respectively, of controls. The number and isoelectric point (pI) values of the isoforms of the alpha 2M from patients with chronic-progressive disease were similar to controls. The average pI of the major form for both groups was 6.6. By contrast, the average pI of the major form from the patients with relapsing-remitting MS was significantly elevated to 7.1, and this group displayed a significantly higher percentage of total recovered activity above pH 7.0. In eleven of the twelve cases examined, the pI of the major form of alpha 2M correctly correlated with the clinical status of the patient. The original clinical diagnosis of the patients was reassessed by a 9-year retrospective interview which verified that 9 of the 10 patients in the follow-up group retained their original clinical diagnosis. These studies demonstrate differential isoform profiles of native alpha 2M from MS patients with progressive versus remitting disease which may be useful in subclassifying MS patients.

Analysis of purified human liver alpha-L-fucosidase by western-blotting with lectins and polyclonal and monoclonal antibodies.

Western-blot analysis [with lectins, polyclonal antibodies (pAbs) and four monoclonal antibodies (mAbs)] was employed to investigate the structural relationship between the separated isoforms and subunits of purified human liver alpha-L-fucosidase. SDS/PAGE and Western-blot analysis indicated the presence of two protein bands of 51 kDa and 56 kDa that were recognized by the pAbs. Polyacrylamide-gel isoelectric focusing (PAG-IEF) followed by blotting indicated that the pAbs and mAbs recognized at least five fucosidase isoforms (pI values 3.6-6.0). Lectin blotting indicated an enrichment of sialic acid residues in the more acidic isoforms. Western-blot analysis indicated that four mAbs recognized the 51 kDa subunit and at least two mAbs recognized the 56 kDa subunit. The subunit composition of the isoforms (separated by PAG-IEF) of human liver alpha-L-fucosidase was investigated by SDS/PAGE. One or two closely spaced bands were found for each isoform with a trend of increasing relative amounts of the high-molecular-mass band in the more acidic isoforms relative to the more neutral isoforms. Neuraminidase treatment of alpha-L-fucosidase resulted in a decrease in the amount of the high-molecular-mass subunit and an increase in the amount of the low-molecular-mass subunit, suggesting that these subunits are related at least in part by sialic acid residues. In addition, blotting with lectins indicated the presence of sialic acid residues only in the high-molecular-mass subunit. N-Glycanase treatment led to the disappearance of the glycosylated 56 kDa and 51 kDa protein bands and the appearance of non-glycosylated protein bands at 48 kDa and 45 kDa. The overall results indicate that (1) N-glycosylation contributes to, but does not account completely for, structural differences in the fucosidase subunits and (2) the more acidic isoforms of fucosidase contain enriched relative amounts of the sialylated high-molecular-mass subunit.

The effect of carbohydrate removal on the properties of human liver alpha-L-fucosidase.

The effect of carbohydrate removal on the properties of the lysosomal enzyme alpha-L-fucosidase has been investigated by comparatively characterizing N-glycanase-treated and mock-treated control fucosidases. N-Glycanase treatment removed approx. 90% of the carbohydrate from purified native human liver fucosidase as determined by carbohydrate assay after gel filtration on Sephadex G-50, and by Western blotting with a lectin-digoxigenin conjugate and densitometric scanning. Removal of carbohydrate from fucosidase does not affect its catalytic activity, its Km value for synthetic substrate, its recognition and rate of hydrolysis of three natural substrates, or its gross conformation as determined by circular dichroism. However, loss of carbohydrate led to significantly decreased activity at acidic pH values (3.1-4.7), a 0.6 pH unit shift to a more neutral optimum and decreased thermostability. The decreased activity at acidic pH values and the more neutral pH optimum of deglycosylated fucosidase suggest that the presence of carbohydrate is physiologically significant in allowing fucosidase to perform its catabolic function more efficiently in the acidic milieu of the lysosome.

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.

The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.

Mammalian alpha-L-fucosidases.

Mammalian alpha-L-fucosidases are a ubiquitous group of relatively large multimeric lysosomal glycosidases involved in the degradation of a diverse group of naturally-occurring fucoglycoconjugates. These enzymes are closely related structurally as indicated by immunochemical cross-reactivity and cloning studies. Mammalian fucosidases are sialoglycoproteins and the carbohydrate, particularly sialic acid, contributes to producing multiple isoforms which can differ in various species as well as in different tissues within a given species. alpha-L-Fucosidases exhibit maximal activity at pH values between 4 and 7, have similar kinetic properties with synthetic substrates (PNP-fucoside and 4-MU-fucoside), and exhibit broad substrate specificity on natural substrates. Numerous linkages (alpha 1-2, alpha 1-3, alpha 1-4, alpha 1-6), primarily to galactose and N-acetylglucosamine, can be hydrolyzed but preference is often seen for small mol. wt water-soluble substrates with fucose in alpha 1-2 linkage to galactose. The importance of alpha-L-fucosidase in mammalian metabolism is evidenced by deficiency or absence of its enzymatic activity leading to a fatal genetic disease, at least in humans and English Springer Spaniels.