Jack A. Alhadeff

Total and lipid-associated serum sialic acid levels in cancer patients with different primary sites and differing degrees of metastatic involvement

Serum total sialic acid (TSA) and lipid‐associated sialic acid (LASA) levels have drawn considerable interest because of carbohydrate aberrations in malignant cells. The current investigation determined the TSA, LASA, total protein (TP), and TSA/TP values for 171 cancer patients with various primary sites and differing degrees of metastatic disease, 102 patients with nonmalignant diseases (pathologic controls), and 42 normal individuals. Data analysis indicated significant (p < 0.01) increases in the mean (±SD) TSA and TSA/TP values in the cancer patients (78.1 ± 19.2 mg/dl and 12.4 ± 3.8 mg/g, respectively) and in the pathologic controls (76.0 ± 7.5 mg/dl and 11.6 ± 2.5 mg/g) when compared to the normal controls (67.3 ± 7.1 mg/dl and 9.0 ± 1.1 mg/g), and a significant decrease in the mean TP values in the cancer patients (6.4 ± 1.1 g/dl) and pathologic controls (6.6 ± 1.1 g/dl) when compared to normal controls (7.5 ± 0.5 g/dl). No significant difference was observed between groups in LASA values. Further analysis of the data in patient subgroups based on the tissue involved, specific disease, or severity of the malignancy indicated that the lack of specificity of the markers was due primarily to restricted subgroups and that the sensitivity of TSA and TSA/TP increased as the malignancy became more severe. The results show that TSA/TP was the most useful of the markers tested for detecting malignancies. This marker should prove useful for monitoring malignant disease recurrence and/or progression and for evaluating the effectiveness of various therapeutic approaches.

Malignant cell glycoproteins and glycolipids

The cell surface is involved in cell growth and division, cell-cell interaction, communication, differentiation and migration, and other processes likely to be involved in malignant transformation and/or the metastatic spread of cancer. Although there are many alterations of glycoproteins and glycolipids on the malignant cell surface, it is unclear whether these alterations are epiphenomena or an integral part of the malignancy process. This article reviews the recent literature and some earlier studies relevant for understanding emerging concepts and trends with respect to malignant cell glycoconjugates. Emphasis is on structural alterations of the carbohydrate portions of malignant cell glycoproteins and glycolipids and on the enzymes (glycosyltransferases and glycosidases) involved in their metabolism. Practical applications derived from malignant cell glycoconjugate studies are discussed briefly with respect to the diagnosis, staging, monitoring, and treatment of malignant disease. The review concludes by indicating which research areas on malignant cell glycoconjugates are likely to be fruitful in increasing our basic understanding of, and ability to deal effectively with, malignant disease.

Mammalian α-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.

Purification and Characterization of Plasma Membrane-Associated Human Sperm α-l-Fucosidase

Abstract Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-l-fucose, have provided further evidence for the plasma membrane association of a novel human sperm, α-l-fucosidase. This α-l-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C24-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated α-l-fucosidase. Both SDS-PAGE and Western blot analysis indicated the α-l-fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis detected the presence of mannose, but no terminal galactose or sialic acid residues. Isoelectric focusing indicated the presence of two major α-l-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of α-l-fucosidase with neuraminidase did not change its isoform profile, providing further evidence for the enzymes lack of sialic acid residues. Kinetic analysis with 4-methylumbelliferyl α-l-fucopyranoside indicated that sperm α-l-fucosidase has a pH optimum near 7, an apparent Km of 0.08 mM, and a Vmax of 6.8 μmol/min/mg protein. The unusual properties of human sperm α-l-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.

Isoenzyme patterns of human liver α-l-fucosidase during development☆

Abstract The pattern of human liver α- l -fucosidase isoenzymes during development has been studied by isoelectric focusing. Seven isoenzymes have been found in livers from fetuses, children and adults. The pattern of isoenzymes appears to change during development. Whereas the most neutral form (I) of α- l -fucosidase is prominent in early fetal development (105–109 days gestation), it is greatly diminished in amount later in fetal development (123–124 days gestation) and in children and adults. Incubation of adult human liver α- l -fucosidase with neuraminidase eliminates the five most acid forms of the enzyme and greatly increases the amount of the most neutral form (I). It is thus possible that sialylation of the most neutral form of α- l -fucosidase accounts, at least in part, for the changes in fucosidase isoenzyme patterns during development.

Analysis of cathepsin D in human breast cancer: Usefulness of the processsed 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 (≤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.

Active-site-directed inactivation of human liver α-l-fucosidase by conduritol C trans-epoxide

Conduritol C trans-epoxide was found to inactivate human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51), exhibiting an apparent dissociation constant of 43 mM. The cis-isomer of the inactivator had no apparent effect on the enzymes activity. The pH profile for the inactivation yielded two apparent pK values of approx. 3.7 and 6.1 alpha-L-Fucose (a competitive inhibitor) was effective in protecting the enzyme from inactivation. These results are consistent with a requirement for two amino acid side chains at the active site involved in the reaction of the enzyme with conduritol C trans-epoxide.

Variant serum β‐hexosaminidase as a biochemical marker of malignancy

Previous studies have demonstrated a variant form of β‐hexosaminidase in metastatic tumor tissue of human liver and in sera from cancer patients with liver metastases. The current investigation examined sera for the presence of the variant form of β‐hexosaminidase from a large and heterogeneous group of cancer patients (with different primary sites and differing degrees of metastatic involvement), from normal controls and pathological controls with nonmalignant diseases. Comparison of the total serum β‐hexo‐saminidase activity levels and the percentage of the total activity comprised of β‐hexosaminidase B (Hex B) revealed no significant differences (P > 0.01) between the three groups. Analytical isoelectric focusing indicated that the variant β‐hexosaminidase was present in 80% of 108 cancer patients, 37% of 27 pathological controls and 11% of 18 normal controls. Examination of subgroups of the cancer patients based on the extent of metastasis revealed that there was a significant increase in total serum β‐hexo‐saminidase activity and the presence of variant β‐hexosaminidase in the sera as the disease progressed. These results suggest that serum β‐hexosaminidase may be a useful marker for monitoring the progression of malignant disease.

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.

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

SummaryCathepsin 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 Mr 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.