John L. Stirling

The polycystin-1 C-type lectin domain binds carbohydrate in a calcium-dependent manner, and interacts with extracellular matrix proteins in vitro.

Mutations in the PKD1 gene are responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). This gene encodes a large membrane associated glycoprotein, polycystin-1, which is predicted to contain a number of extracellular protein motifs, including a C-type lectin domain between amino acids 403--532. We have cloned and expressed the PKD1 C-type lectin domain, and have demonstrated that it binds carbohydrate matrices in vitro, and that Ca(2+) is required for this interaction. This domain also binds to collagens type I, II and IV in vitro. This binding is greatly enhanced in the presence of Ca(2+) and can be inhibited by soluble carbohydrates such as 2-deoxyglucose and dextran. These results suggest that polycystin-1 may be involved in protein-carbohydrate interactions in vivo. The data presented indicate that there may a direct interaction between the PKD1 gene product and an ubiquitous extracellular matrix (ECM) protein.

β N-acetylhexosaminidases A and S have similar sub-cellular distributions in HL-60 cells

Abstract In HL-60 cells the most abundant isoenzymes of β-N-acetyl-hexosaminidase are A (ap) and S (αα ). Sub-cellular fractionation of HL-60 cells by differential centrifugation showed that both A and S forms were present in the lysosomal and post-lysosomal (soluble) fractions in approximately equal abundance. Ion-exchange chromatography showed that a fraction enriched with plasma membranes had the A form, and a form of β-N-acetylhexosaminidase less acidic than A, but there was no S. Analysis of the α-subunits of β-N-acetylhexosaminidases A and S using Western blotting and immuno-detection with antisera raised to synthetic peptides showed that mature α-subunits were present in both A and S isolated from the lysosomal fraction. This observation establishes that the αα-dimer of β-N-acetyl-hexosaminidase (S) can be transported to lysosomes in HL-60 cells whereas there is good evidence that this does not take place in fibroblasts. HL-60 cells were not stimulated to secrete lysosomal enzymes by incubating them with NH4C1 and, unlike fibroblasts, are unlikely to use mannose-6-phosphate mediated transport of β-N-acetylhexosaminidases to lysosomes. Comparison of the sequence of the β-N-acetylhexosaminidase a-subunit with a 43 amino acid sequence of cathepsin D, thought to function in the mannose-6-phosphate independent targeting of this enzyme to lysosomes, showed alignment in a region towards the C-terminus in which 21% of the residues were identical with the interposition of a one amino acid gap.

Evidence for the regulation of β-N-acetylhexosaminidase expression during pregnancy in the rat.

It is believed that the lysosomal glycohydrolase beta-N-acetylhexosaminidase plays a part in several important processes of reproduction and it has been postulated that this enzyme is subject to hormonal regulation. During pregnancy, activity levels of the enzyme are strongly increased in both human and rat serum. However, little is known about the expression of this enzyme in the female reproductive apparatus and there is no evidence linking the production of hexosaminidase alpha- and beta-subunits to pregnancy. To clarify these aspects better, we examined the enzyme activity, isoenzyme subunit composition and distribution, as well as steady state levels of alpha- and beta-subunit mRNAs in the female reproductive organs and in other selected tissues of pregnant and non-pregnant rats. Among the female rat tissues tested, the ovary and kidney had the highest specific activity. Pregnancy modulated the hexosaminidase activity differently in the tissues examined. In pregnant rats, the activity decreased in the ovary but increased significantly in the uterus, liver and to a lesser extent in other tissues. The decreased hexosaminidase activity in the ovary from pregnant rats appeared to be accompanied by a disproportionately large decrease in beta-subunit mRNA abundance, whereas in the uterus and liver, an increased abundance of this transcript was detectable. The abundance of alpha-subunit mRNA was comparable in pregnant and control rat tissues. Hexosaminidase histochemical staining of tissue sections clearly demonstrates that the greatly increased activity of hexosaminidase in the uterus during pregnancy is largely due to the enzyme in the endometrium, and not to the uterus as a whole. The overall results provide evidence that, during pregnancy, a mechanism(s) of regulation of beta-N-acetylhexosaminidase expression is in operation, and that the enzyme is differentially regulated in rat tissues.

Localization of the pro-sequence within the total deduced primary structure of human β-hexosaminidase B

The β subunit of β‐hexosaminidase (β‐N‐acetylhexosaminidase, EC 3.2.1.52) is synthesized in the rough endoplasmic reticulum as a prepropolypeptide. After the loss of the signal peptide and formation of an enzymatically active dimer, the pro‐enzyme is either secreted from the cell or transported into the lysosome for processing to its mature form. In order to characterize the early posttranslational events we have purified nearly 1 mg of pro‐hexosaminidase B from the NH4Cl containing medium of fibroblasts derived from a patient with the infantile form of Tay‐Sachs disease. The partial N‐terminal sequence was mapped to a position 42 residues C‐terminal to the first in‐frame ATG (Met residue) and 79 residues N‐terminal to the known mature N‐terminus. This position corresponds to that predicted for the cleavage of a 17 amino acid signal peptide generated through the use of the third rather than the first in‐frame ATG as the initiation site for protein synthesis.

β-N-Acetylhexosaminidases in the spleen of a patient with hairy-cell leukaemia

Abstract The spleen from a patient with hairy-cell leukaemia had β - N -acetylhexosaminidase activity that could be resolved into three isoenzymes by chromatography on phenyl boronate agarose. Two of these were the major forms, A and B, found in normal tissues but, in addition, there was an ‘extra’ form that accounted for 15% of total activity. The ‘extra’ form hydrolysed the synthetic substrate 4-methylumbelliferyl- β - N -acetylglucosamine 6-sulphate, indicating the presence of α-subunits. It was more acidic than A, was less heat-stable and showed no generation of B on denaturation under a variety of conditions. These findings and the immunoblot (Western blotting) analysis demonstrate that the ‘extra’ form is entirely composed of α-subunits, and most closely resembles S, the residual activity in Sandhoffs disease.

Mouse beta-mannosidase: cDNA cloning, expression, and chromosomal localization

Abstractβ-mannosidase is an exoglycosidase involved in the degradation of N-linked oligosacharides moieties of glycoproteins. Lack of β-mannosidase activity leads to the lysosomal disorder β-mannosidosis (MIM 248510). We have isolated and sequenced the gene encoding the mouse β-mannosidase. Comparison of the deduced amino acid sequence of mouse, human, bovine, and goat β-mannosidase showed 64% identity, reflecting a high degree of evolutionary conservation. Analysis of a multiple tissue northern blotting revealed a major transcript of about 3.7 kb in all tissues examined. The northern analysis also demonstrates that there is differential tissue mRNA expression. The mouse β-mannosidase gene (Bmn) was mapped to the distal end of Chromosome (Chr) 3, in a region that is homologous with a segment of human Chr 4 containing the orthologous human gene.

Promoter characterization and structure of the gene encoding mouse lysosomal alpha-d-mannosidase

Abstract. Mouse lysosomal α-d-mannosidase (EC 3.2.1.24) is an enzyme involved in the catabolism of N-linked glycoproteins. The gene is differentially expressed in mouse tissues, and the highest level of mRNA is found in the epididymis. The expression of mannosidase in the epididymis may be hormonally regulated, since its activity increases with age. To understand the factors affecting the expression of mouse mannosidase, we isolated and characterized the promoter and determined the exon-intron structure. The gene is about 15 kb, consists of 24 exons, and the 5′ flanking region contains GC-rich regions, TATA boxes, CAAT boxes, and putative binding sites for the transcription factors Sp1, AP2, and PEA3. PEA3 factor may participate in the transcriptional control of mannosidase expression in the mouse epididymis. In fact, it has been demonstrated that the PEA3 motif is spatially and temporally expressed within the mouse epididymis, and its accumulation is controlled by androgens and testicular factors. A 1279-bp fragment from the initiation codon had the strongest promoter activity, and three different transcription start sites were identified at positions -131, -149, and -174.

Structural organization and expression of the gene for the mouse GM2 activator protein

The GM2 activator protein is an essential component for the degradation of GM2 ganglioside by hexosaminidase A in vivo. Mutations in the human gene coding for the GM2 activator protein cause the AB variant of GM2-gangliosidosis, a condition that is clinically indistinguishable from Tay-Sachs disease. To understand better factors affecting the expression of the GM2 activator protein gene (Gm2a) in mouse tissues, we have determined its exon-intron organization and analyzed its promoter region.Gm2a is about 14 kb, has four exons, and the 5′ flanking region contains a CAAT box, Spl binding sites, AP-1, AP-2 sites, and a pair of IRE sites. A 1.2-kb fragment upstream from the initiation codon was shown to have promoter activity in NIH 3T3 cells. Similarities between the elements present in Gm2a and Hexa promoters might in part explain their similar expression patterns in mouse tissues. The different levels of GM2 activator protein mRNA in liver, kidney, brain, and testis are not owing to the use of different transcription start sites, because a single start site was found 50 bp upstream from the initiation codon in each these tissues. Northern blot analysis demonstrated variation in the GM2 activator protein mRNA expression during mouse development. Gm2a was mapped to Chromosome (Chr) 11, where it co-segregated with Csfgm.

Distribution of active α- and β-subunits of β-N-acetylhexosaminidase as a function of leukaemic cell types

Abstract β - N -Acetylhexosaminidase isoenzymes, and the distribution of the α - and β -subunits forming the enzyme in a representative series of fresh leukaemic cells and in established leukaemic cell lines, were obtained by using a combination of DEAE-cellulose chromatography and assay with the fluorogenic substrates 4-methylumbelliferyl- β - N -acetylglucosaminide hydrolyzed by both α and β subunits, and 4-methylumbelliferyl- β - N -acetylglucosaminide-6-SO 4 hydrolyzed only by hexosaminidase isoenzymes containing α -subunits. The presence of hexosaminidase S (the α α dimer), was found in all the leukaemic cell populations we surveyed, but not in normal human cells. The presence of this isoenzyme can therefore be considered as an additional marker of leukaemic cells. A prevalence of hexosaminidase A and A-like intermediate forms ( α β structure), characterize leukaemic cells of myeloid origin, whereas greater amounts of hexosaminidase B and B-like intermediate forms ( β β structure), were consistent attributes of leukaemic cells of lymphoid origin. An over-expression of β -subunits in blasts might be related to their undifferentiated status. These changes in the isoenzymes of hexosaminidase may prove informative about a variety of changes in the biology of leukaemic cells that could range from chromosomal alterations to changes in the proteolytic processing and glycosylation.

Discovery of the hexosaminidase isoenzymes

Publisher Summary This chapter deals with the discovery of the Hexosaminidase Isoenzymes. The definition of the clinical symptoms and the realization that this was a familial disorder, long preceded the laboratory analytical technology that was necessary for a precise identification of the biochemical lesion. As precise assay of enzyme activity on these hydrophobic or amphipathic natural substrates was an exacting task necessitating the preparation and use of radio-labelled glycolipids, many workers elected to study the enzymology of mammalian glycosidases using methods based on synthetic water-soluble substrates. This chapter illustrates how skill and perseverance with these natural substrates led to the discovery of such an activating factor and also reveals, at a very early stage, the remarkable degree of genetic heterogeneity that could arise in what turned out to be a family of defects. The effect could be attributed to Hexosaminidase A being a multisialated version of Hexosaminidase B. The ultimate reduction of electrophoretic mobility to that of Hexosaminidase B and the transient appearance of a B-like species during the heat denaturation of A, supported a very close structural relationship.