alpha1,3 Fucosyltransferase, alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, and Le(x) glycoconjugates in developing rat brain.

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Le(x)) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15-P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10-P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an alpha1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5-10) with different types of acceptors, and the differential expression of Le(x) containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, alpha-L-fucosidase, beta-D-galactosidase and alpha-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases.

The rat alpha1, 3-fucosyltransferase (rFucT-IV) gene encodes both long and short forms of the enzyme which share the same intracellular location.

Fucosyltransferase (FucT) activity has been detected on the surface of mouse germ cells and rat Sertoli cells, and has been postulated to play a role in cell-cell interactions. A recently cloned rat FucT (rFucT-IV) is expressed in the testes, and thus is a candidate for encoding the cell-surface FucT activity. This study maps the 5'-ends of several rFuc-T-IV mRNAs, and these results suggest that initiation of transcription may occur both upstream of the first ATG, as well as between the first two closely spaced, in-frame ATGs. Thus, in certain tissues, notably spleen, significant amounts of both a long and a short form of rFucT-IV would be predicted. This study also determines some basic properties of both the long and short forms of rFucT-IV, and investigates whether the use of alternative ATGs would allow FucT activity to be expressed both on the cell surface and in the Golgi. Plasmids that encode FLAG-epitope-labeled rFucT-IVs that initiate from either of the two ATGs were constructed, and rFucT-IV was expressed either in vitro using cell-free rabbit reticulocyte lysate, or after transfection in tissue culture. The results from these studies demonstrate that rFucT-IV is a glycosylated, transmembrane protein with a short cytoplasmic tail, and that either of the two ATGs in the 5' region of the rFucT-IV gene are capable of acting as functional initiators of translation in vitro, to produce enzymatically active glycoproteins. However, no difference in the intracellular localization between the transferase containing a 48 amino acid or a 15 amino acid cytoplasmic tail was detected by immunocytochemistry, as both show the same pattern of Golgi-like staining in several different cell types, with no indication of surface expression. Thus, the additional amino-terminal 33 amino acids of the long form of rFucT-IV do not appear to influence its intracellular location in the cell types investigated.

Engineered complementation in Escherichia coli aspartate transcarbamoylase. Heterotropic regulation by quaternary structure stabilization.

Escherichia coli aspartate transcarbamoylase regulates pyrimidine biosynthesis by altering its activity homotropically in response to one of its substrates and heterotropically in response to nucleotide effectors. The mechanism of this regulation involves two structurally and functionally different forms of the enzyme, one with low activity and low affinity for substrates (T state) and the other with high activity and high affinity for substrates (R state). Heterotropic regulation may be due to the direct transmission of a regulatory "signal" between the regulatory site and the active site some 60 A away and/or may involve altering the relative stability of the two forms of the enzyme. By combining a T state-stabilized mutant version of the enzyme, previously thought to have a defect in a heterotropic transmission pathway, with a known R state-stabilized mutant enzyme, we were able to restore some properties of the wild-type enzyme. These data imply that the relative stabilization of the T and R states of the enzyme is in part responsible for the homotropic and heterotropic properties of aspartate transcarbamoylase and that direct pathways for transmission of the heterotropic signals are unlikely.

Comparison of promoter strengths on gene delivery into mammalian brain cells using AAV vectors.

Recent reports have suggested that delivery of genes flanked by AAV ITRs may be useful for gene therapy of diseases that involve the brain. We have compared the efficiency of gene expression in vitro in CNS-derived cells from four different promoters when the transgene is flanked by AAV ITRs, using both transfection via cationic liposomes, and infection via rAAV. The human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, and the chicken beta-actin promoter coupled to the CMV enhancer were able to drive expression of the reporter gene beta-galactosidase in all tumor and primary brain cell cultures tested. Although the relative order of efficiency differed between cell types, the CMV promoter was always the strongest, generally by at least one order of magnitude. A comparison of the relative levels of expression seen between different cell types on transfection and infection suggest that not all CNS-derived cells are infected equally efficiently by rAAVs. High level of expression were seen within 24 h of transgene delivery by either transfection or infection, dropping dramatically within days. All cell types and promoters showed the same decline, suggesting that transient expression by rep-rAAVs may be efficient, but stable expression as detected in this system is a low frequency event. In vivo studies using the CMV promoter also suggest that although rep-rAAVs are able to infect efficiently CNS cells and produce high levels of gene expression shortly after transduction, the majority of such infections do not lead to stable high-level expression of transgenes.

Overexpression and purification of the trimeric aspartate transcarbamoylase from Bacillus subtilis.

A procedure has been developed for the overexpression and purification of milligram quantities of the Bacillus subtilis aspartate transcarbamoylase. The plasmid pEK171, carrying the B. subtilis pyrB structural gene under the control of the Escherichia coli pyrBI promoter, was transformed into the E. coli strain EK1104 and the enzyme overexpressed to approximately 50% of total soluble protein under extreme derepression of the pyrimidine pathway. The enzyme was subsequently purified by means of ammonium sulfate fractionation, anionic exchange chromatography using Q-Sepharose Fast Flow resin, negative chromatography on Matrex Gel Red A agarose, and hydrophobic interaction chromatography using Matrex Phenyl Cellufine. The purification yields approximately 60 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties very similar to those of the enzyme purified from B. subtilis cells.