Further evidence of human αa-l-fucosidase polymorphism

The results of a comparative study of multiple forms of α-l-fucosidase from human kidney, liver, placenta and blood serum provide evidence for α-l-fucosidase polymorphism. On the basis of the data obtained the existence of certain phenotypic groups of α-l-fucosidase, dissimilar both in the quantitative and qualitative composition of the enzyme's multiple forms, was revealed. Some possible reasons for α-l-fucosidase polymorphism are discussed. It is suggested that the data on α-l-fucosidase polymorphism may be used in diagnosis and in the isolation and preparation of individual enzyme forms.     

Human liver alpha-L-fucosidases

Normal human liver α-l-fucosidase can be separated by ion-exchange chromatography into two forms. These are also separated by gel permeation chromatography on Sephadex G-200. Fucosidase I is a macromolecular form excluded from Sephadex G-200 and adsorbed on DEAE-cellulose at pH 6.5. Fucosidase II has a low molecular weight (50000) and is not adsorbed under these conditions. The two enzymes have the same pH optima in the region of 5.0 but have distinctive pH activity profiles due to the relative heat instability of fucosidase II. The results bear a close resemblance to the pig kidney enzymes previously reported.     

Fluorescent assay of alpha-L-fucosidase

Human α-l-fucosidase can be assayed using the fluorigenic substrate 4-methylumbelliferyl α-l-fucoside. This substrate is hydrolysed more readily than the p-nitrophenyl derivative by the human liver enzyme but the serum enzyme shows approximately the same activity towards the latter substrate. The pH profiles are similar with both substrates with a slight shift in the maxima and both iso-enzymic forms of α-l-fucosidase are detected by either substrate. The greatly increased sensitivity of assay by the fluorescence method favours its use in diagnostic studies on fucosidosis.     

Electrophoretic forms of human liver alpha-L-fucosidase and their relationship to fucosidosis (mucopolysaccharidosis F)

1. Human liver α-L-fucosidase was studied by starch gel electrophoresis and isoelectric focusing both before and after treatment with neuraminidase. These studies revealed the presence of 5–6 bands on starch gels and six isoelectric forms with pI's of 6.9, 6.5, 6.1, 5.9, 5.7 and 5.5. The activity of the two most acidic forms was reduced after neuraminidase treatment.2. Liver tissue from a patient with fucosidosis had approximately 4% of normal α-l-fucosidase activity. Starch gel electrophoresis revealed that all electrophoretic forms of the enzyme appear to be deficient.3. Apparent Michaelis constants (K_m 's) determined for both the normal and fucosidotic liver α-l-fucosidase for 4-methylumbelliferyl-α-l-fucopyranoside were found to be 0.085 mM and 0.058 mM, respectively.4. The pH activity profile of normal and fucosidotic liver α-l-fucosidase were very similar with major pH optima at 5.4 and 5.3, respectively.     

A sensitive fluorometric assay for alpha-KL-fucosidase.

A sensitive fluorometric assay for α-l-fucosidase applicable to serum or plasma is described. Some normal individuals have been found to have an extremely low level of α-l-fucosidase. This low activity enzyme has an altered pH versus activity profile but a K_m of the same order of magnitude as the high activity enzyme.     

α-l-Fucosidase m cystic fibrosis: analysis of skin fibroblasts and liver

The lysosomal enzyme α-l-fucosidase has been examined by thin layer gel and column isoelectric focusing in skin fibroblasts and liver from patients with cystic fibrosis and controls. All three common phenotypes of the enzyme were observed in both control and CF fibroblasts. When individuals of the same α-l-fucosidase phenotype were compared, no major differences between the isozyme profiles of cystic fibrosis patients and controls were detected in either fibroblasts or liver tissue.     

Isozymes of human alpha-L-fucosidase detectable by starch gel electrophoresis

Methods are described for the electrophoresis in starch-gel of human α-l-fucosidase and for the detection of the enzyme using the fluorogenic substrate 4-methylumbelliferyl-α-l-fucopyranoside. The electrophoretic pattern of the enzyme was examined in leucocytes, serum, cultured fibroblasts and long-term lymphoid cell lines. The enzyme from cultured lymphoid cell lines was found to consist of up to 6 clearly resolved electrophoretic isozymes plus a diffuse, more anodal region of lower staining intensity. The enzyme from leucocytes and cultured skin fibroblasts was less clearly resolved, but these cell types appeared to have components corresponding in mobility to the isozymes of lymphoid lines. In contrast, the enzyme from serum (or plasma) showed only a diffuse region of activity, with an anodal mobility slightly greater than that of the most anodal lymphoid line isozymes. Evidence is presented which indicates that the electrophoretic heterogeneity of α-l-fucosidase is due in part to the binding of sialic acid to the primary gene product. None of the isozymes was detectable in lymphoid cell lines, serum or cultured fibroblasts from a patient with fucosidosis, an inborn error of metabolism.     

Susceptibility to neuraminidase of α-l-fucosidase and N-acetyl-β-d-glucosaminidase of cystic fibrosis, I-cell and neuraminidase-deficient fibroblasts

Intracellular α-l-fucosidase and hexosaminidase showed similar isoelectrofocusing patterns in control, cystic fibrosis and neuraminidase-deficient fibroblasts and were unaffected by neuraminidase treatment. An I-cell strain excreted these two enzymes at 3–4 times the rate of the three other cell types. I-cell and neuraminidase-deficient cells excreted more of the electronegative forms of these enzymes than control and cystic fibrosis cells. Extracellular hexosaminidase A and B were both sensitive to neuraminidase for the four cell types. Extracellular α-l-fucosidase consisted of a pH 6.1 form insensitive to neuraminidase and other forms that were sensitive and changed to a pI 7.0–7.1 form. Cystic fibrosis extracellular a-l-fucosidase and hexosaminidase behaved as for control fibroblasts.     

Lysosomal enzyme activities in cultured lymphoid cell lines

Several lysosomal enzyme activities in cultured lymphoid cell lines were studied during 3 phases of cell culture; logarithmic growth phase, stationary phase and decline phase.Enzyme induction during cell growth was found in N-acetyl-hexosaminidase, β-galactosidase and α-l-fucosidase, but no induction in a-d-mannosidase, α-glucosidase and β-glucuronidase. The latter two enzymes were unchanged during all cell culture phases.A drop in α-l-fucosidase and α-d-mannosidase activity was found during the stationary and decline phases of cell culture.     

Cystic fibrosis serum α-l-fucosidase: Confirmation of normal activity levels and normal kinetic and isoelectric focusing properties

Comparable α-l-fucosidase activity was found for normal and cystic fibrosis sera, 7.8 ± 3.3 and 9.8 ± 4.7 nmol/min/ml, respectively. Isoelectric focusing of normal and cystic fibrosis sera α-l-fucosidase revealed similar isoelectric profiles with most activity between isoelectric points of 4.7–5.4. Kinetic analysis of normal and cystic fibrosis sera α-l-fucosidase revealed identical apparent Michaelis constants for the 4-methylumbelliferyl substrate (51 ± 6 μmol/l and 50 ± 8 μmol/1, respectively), similar pH optima curves (both have broad optima between pH 4.8 and 6.5) and identical thermostability curves at three different preincubation temperatures (37°, 45° and 55°C).     

N-Acetyl β-d-glucosaminidase and α-l-fucosidase activities in relation to glycosylated hemoglobin levels and to retinopathy in diabetes

N-Acetyl β-d-glucosaminidase and α-l-fucosidase were determined in human sera from 25 control subjects, in 23 diabetic patients without retinopathy and in 22 diabetic patients with retinopathy.The results show significantly higher N-acetyl β-d-glucosaminidase activity in diabetic patients independently of the development of retinopathy and also independently of the length of diabetes. No correlation was found between either serum enzymes and serum glucose concentration and glycosylated hemoglobin (HbA,).     

Cobalamin R binder as a possible model molecule for glycoprotein study in cystic fibrosis

The isoprotein pattern of semi-purified R binder (an acidic glycoprotein which binds cobalamin) from saliva and sera of 8 cystic fibrosis patients was compared to that of R binder from samples of 5 healthy children. In cases of cystic fibrosis, the mean isoelectric point of salivary R binder was increased from 3.78 up to 4.34 and its microheterogeneity was reduced. These significant physicochemical modifications were not observed with R binder from cystic fibrosis sera and they did not correlate with the β-galactosidase, α-mannosidase, α-l-fucosidase nor neuraminidase activity of saliva. We propose the R binder as a model molecule to study the glycoprotein metabolism in cystic fibrosis since it contains 30–40% carbohydrate, is easily complexed with cyano[⁵⁷Co]cobalamin and is present in most tissues and fluids of the human organism.     

Acid hydrolase deficiencies and abnormal glycoproteins in mucolipidosis. 3 (pseudo-Hurler polydystrophy)

Biochemical studies were performed on two siblings with pseudo-Hurler polydystrophy (ML III). Although mucopolysaccharide excretion was not in excess, the levels tended toward the upper limit of the normal range. Electrophoretic studies revealed abnormal glycoprotein components in the urine of both patients. Leukocyte extracts had a higher than normal content of neutral sugar, sialic acid and hexosamine, suggesting the presence of excess glycoprotein(s). The most striking abnormalities were in the acid hydrolase activities of cultured fibroblasts. Affected cells had essentially no detectable α-l-fucosidase, and the activities of seven other glycosidases ranged from only 11 to 37% of normal controls. The acid phosphatase activity in the fibroblast cultures was normal, while β-glucosidase activity was more than twice the mean level in normal control cultures. Concomitant with the marked decrease in cellular activities of eight of the ten acid hydrolases studied, an excess of nearly all of these enzymes was found in the culture medium of the ML III patient. The similarities in biochemical and other abnormalities in ML III to those reported for ML II (I-cell disease) are discussed.     

Alpha-L-fucosidase and alpha-D-mannosidase activity in the white blood cells in the disease and carrier state of fucosidosis

α-l-Fucosidase and α-d-mannosidase activity in white blood cells from a patient with fucosidosis and her family members were studied.α-l-Fucosidase activity of the patient was completely lacking, whereas the α-l-mannosidase activity was increased and relative thermostability of this enzyme was also observed. Apparent K_m value of α-d-mannosidase was similar in the patient and the control. When the ratio of α-l-fucosidase to α-d-mannosidase activity was calculated, the value of the parents was intermediate between that of the patient and the control.     

Adenovirus-Transduced Lung as a Portal for Delivering α-Galactosidase A into Systemic Circulation for Fabry Disease

Gene therapy efforts have focused primarily on the use of either the liver or skeletal muscle as depot organs for the production of a variety of therapeutic proteins that act systemically. Here we examined the lung to determine whether it could function as yet another portal for the secretion of proteins into the circulation. Fabry disease is caused by a deficiency of the lysosomal hydrolase α-galactosidase A, resulting in the abnormal deposition of the glycosphingolipid globotriaosylceramide (GL-3) in vascular lysosomes. Pulmonary instillation of a recombinant adenoviral vector (Ad2/CMVHI-αgal) encoding human α-galactosidase A into Fabry mice resulted in high-level transduction and expression of the enzyme in the lung. Importantly, enzymatic activity was also detected in the plasma, liver, spleen, heart, and kidneys of the Fabry mice. The detection of enzymatic activity outside of the lung, along with the finding that viral DNA was limited to the lung, indicates that the enzyme crossed the air/blood barrier, entered the systemic circulation, and was internalized by the distal visceral organs. The levels of α-galactosidase A attained in these tissues were sufficient to reduce GL-3 to basal levels in the lung, liver, and spleen and to ∼ 50% of untreated levels in the heart. Together, these results suggest that the lung may be a viable alternate depot organ for the production and systemic secretion of α-galactosidase A for Fabry disease.     

Glyoxalase enzyme system in human muscular dystrophy.

Glyoxalase I and glyoxalase II activities were determined in skeletal muscle of control subjects and of patients with Duchenne dystrophy, other major forms of muscular dystrophies and certain neuromuscular disorders. The glyoxalase I activity was normal in all diseases examined except in Duchenne and limb girdle types of muscular dystrophy, where it showed a significant moderate decrease. The glyoxalase II activity in normal human muscle was very low, and the activity was unaltered in muscle of patients with Duchenne and other major forms of muscular dystrophies and spinal muscular atrophy. The selective decrease of glyoxalase I activity in recessively inherited muscular dystrophies may have some relevance to some phases of these disease processes.     

METABOLISM OF S AND R FORMS OF PNEUMOCOCCUS

In the present paper are given the results of studies on the respiratory and glycolytic metabolism of Pneumococcus Types I, II, and III, and of the R forms derived from these. The metabolism of the S and R forms are compared, and the relationship between changes in virulence, changes in chemical constitution, and changes in metabolism is discussed. 1. There is no respiration in Ringer solution unless sugar is added. 2. The pH (7.8) that is optimal for growth of pneumococcus is also the pH at which the maximum respiration occurs. 3. The intensity of respiration varies with type in the strains used. The respiratory capacity of Type I is 56 per cent of that of Type III, which in turn is 71 per cent of that of Type II. 4. The anaerobic glycolysis is approximately the same for all three groups. 5. Pneumococcus Type I is capable of aerobic glycolysis. 6. Pneumococcus Types II and III do not effect glycolysis aerobically. 7. The energy set free in respiration is considerably greater than that set free in glycolysis. 8. The oxidation quotient for lactic acid is of the same order as found by Meyerhof in muscle and by Warburg for mammalian tissues. 9. The respiratory capacities of Types I and III are changed on conversion of the smooth to the rough form. (a) For Type I the respiration is increased 110 per cent. (b) For Type III the respiration is diminished 45 per cent. (c) For Type II there is only a slight diminution in respiratory activity (16 per cent). 10. The anaerobic glycolysis is increased about 25 per cent on the average for all R forms irrespective of type derivation. 11. Type I on being converted to the R form, loses its capacity for aerobic glycolysis. 12. Type III, on being converted to the R form, gains the capacity for aerobic glycolysis. 13. The oxygen consumption by Pneumococcus compared with that of the human tubercle bacillus and of mammalian tissue, for the same time intervals, weight for weight, is as follows: (a) Pneumococcus Type I consumes thirteen times as much oxygen as does the tubercle bacillus (H37). (b) Pneumococcus Type II consumes thirty-four times as much oxygen as does the tubercle bacillus. (c) Pneumococcus Type II consumes over twenty times as much oxygen as does isolated rat kidney tissue, and almost 100 times as much oxygen as does isolated dog muscle.     

Creatine kinase forms in human skeletal and cardiac muscle.

The creatine kinase (CK) activity in human skeletal and cardiac muscle submitted to QAE-Sephadex chromatography was found to distribute into three peaks (m and h I, II, III fractions). Characterization according to electrophoretic behaviour, approximate molecular weight, thermal stability and immunological properties unambiguously demonstrated the coincidence of fractions I and III with reference MM-CK and MB-CK isoenzymes, while both cardiac and muscular forms II proved to escape any assignment within the dimeric (M, B) model. A higher molecular weight than for reference CK and the absence of interaction with BB-antiserum resulted for both forms II, which on the contrary were found to diverge as for reactivity to MM-antiserum and stability characteristics. Quantitation of the CK forms, attempted on a limited number of samples, confirmed the expected relative levels of MM-CK and BB-CK and gave evidence for the presence of hII- or mII-CK amounting up to 30% of the total activity in cardiac and skeletal muscle, respectively. The results of the study both confirm a complexity greater than supposed for the CK system and point to the inadequacy of several of the commonly used analytical approaches to derive correct information.     

Comparison of the urinary glycoconjugates excreted by patients with type I and type II fucosidosis

Glycoconjugates were isolated, by repeated chromatography on Biogel P-4, from urine of two patients with different phenotypes of fucosidosis, type I (acute) and type II (chronic). Studies of the isolated compounds with thin-layer chromatography, chromatography on Biogel P-4, gas-liquid chromatography, and 1H- and 13C-nuclear magnetic resonance spectroscopy showed differences in the excretion patterns of fucosyl glycoconjugates in the urine of these patients. The amount of the diglycosylasparagine, fuc alpha 1-6glcNAc-Asn, excreted was significantly lower for the type I than for the type II patient. On the other hand, the reducing hexasaccharide gal beta 1-4(fuc alpha 1-3)glcNAc beta 1-2man alpha 1-3/6man beta 1-4glcNAc was present in much greater quantities in urine from the type I patient. The differences in the excretion patterns of these glycoconjugates may be attributed to different substrate specificities for the residual alpha-L-fucosidases in the two forms of the disease. I propose that such differences may be exploited for the early laboratory diagnosis of the type II form of the disease, particularly by thin-layer chromatography.