Jack W. Rip

Distribution, metabolism and function of dolichol and polyprenols

Polyisoprenoid alcohols consisting of 9 or more isoprene units are present in all living cells. They can be fully unsaturated (polyprenols) or alpha-saturated (dolichol). Dolichol forms may have additional saturation at or near the omega-end. Some species contain ony dolichol or only polyprenols while others have nearly equal amounts of both types. Some polyisoprenoid alcohols consist entirely of trans isoprene units but most, including dolichol, contain both trans and cis units. Considerable advances in lipid methodology have occurred since the first review of polyisoprenoid alcohols by Hemming in 1974. For example, direct analysis of both dolichol and Dol-P by HPLC has replaced earlier methods which were often both insensitive and inaccurate. The availability of radiolabeled dolichol and polyprenols has facilitated studies concerning the metabolism and distribution of these compounds. Those studies suggest that only a small portion of the dolichol present in cells is likely to be involved in glycosylation. Polyisoprenoid alcohols are usually present at a family of homologues where each differs in size by one isoprene unit. Little or no size related specificity has been observed for any reaction involving dolichol or polyisoprenol intermediates. The overall length of polyisoprenoid alcohols may, however, affect the manner in which these compounds influence the physical and biochemical properties of membranes. Studies on the biosynthetic pathway leading from cis, trans Pol-PP by phosphatase action. The formation of the dolichol backbone from a polyprenol requires the action of an additional enzyme, an alpha-saturase. This enzyme does not always act at the level of a single common substrate, since Pol-PP, Pol-P, and polyprenol all appear to be utilized as substrates. The major product of the de novo pathway differs among different species. Dol-P would appear to be the most energy efficient end-product since it can participate directly in glycoprotein formation. Most often, however, Dol-P is not the major product of metabolic labeling experiments. In some cases, dolichol is formed so that rephosphorylation is required to provide Dol-P for participation in glycoprotein formation. The kinase responsible for this phosphorylation appears to bypass the considerable stores of dolichol present in tissues (i.e. sea urchin eggs) in favor of dolichol derived directly from de novo synthesis. Although HMGR is a major regulatory component of the pathway leading to polyisoprenoid alcohols and cholesterol, control is most often not co-ordinated.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct estimation of dolichyl phosphate in rat liver by high pressure liquid chromatography.

A method involving reverse-phase high pressure liquid chromatography has been developed for determining the concentration of dolichyl phosphate (Dol-P) in tissues. Individual Dol-P homologs are resolved and amounts as small as 50 ng can be detected. Rat liver was found to contain 2.4 μg Dol-P/g wet weight, or ca. 4% of total liver dolichol. In contrast, rat liver microsomes contained 64 ng Dol-P/mg protein, which is about 40% of total microsomal dolichol. This enrichment in Dol-P is consistent with the role of microsomes as the major site of Dol-P-mediated, glycoprotein biosynthesis in liver

Differences in polyisoprenoid alcohols of mono- and dicotyledonous seeds

A lipid fraction enriched in polyisoprenoid alcohols was prepared from seeds of a number of crop plants, using Florisil chromatography. Analysis by HPLC of the fraction from soybeans showed a series of peaks corresponding to α-saturated homologues (dolichols) from 15 to 22 isoprene units in length. Similar results were obtained with seeds of other dicotyledonous species (rapeseed, peanuts, mung beans, navy beans and peas). In contrast, analysis of the seeds of monocotyledonous plants (wheat, rye, barley, rice and corn) by HPLC gave split peaks, indicating the presence of nearly equal amounts of 2 different homologous series of compounds. The polyisoprenoid material isolated from wheat germ was subsequently shown to consist of a mixture of dolichols and α-unsaturated homologues (polyprenols). Treatment with managanese dioxide selectively oxidized the polyprenols to the corresponding aldehydes, which were separated from the dolichols by TLC. The identity of the components was established by infrared-nuclear magnetic resonance (IR-NMR) spectroscopy and by comparison with authentic standards on high performance liquid chromatography (HPLC). The concentration of polyisoprenoid alcohols in seeds varied from 1–16 mg/100 g. Seeds of different species showed some differences in the pattern of homologues present.

Age-associated changes in dolichol and dolichyl phosphate metabolism in the kidneys and liver of mice

The total amounts of cholesterol, dolichol and dolichyl phosphate (Dol-P) in kidneys and liver from 1- and 24-month-old mice were measured after saponification of the tissues. The cholesterol content of kidneys decreased by about 4-fold per g of tissue between the two ages, while the amount of cholesterol/g liver remained constant. The dolichol content of both tissues increased by about 2-fold. The amount of Dol-P in kidneys increased by 7-fold over 23 months, while the concentration in liver decreased some 6-fold. Metabolic labelling experiments using tritiated water demonstrated that the rate of synthesis of total dolichol (dolichol + Dol-P) in the kidneys did not change as a function of age. However, the kidneys of young mice incorporated 3-fold more radioactivity into dolichol than Dol-P, whereas this distribution was reversed in old mice. In liver, the rate of dolichol synthesis decreased 2-fold over 23 months. It was not possible to compare the rates of Dol-P biosynthesis between ages as Dol-P was undetectable in liver of old mice. The relative abundance of the 19 isoprene unit homologs of both dolichol and Dol-P decreased approx. 5% while the 17 isoprene unit homologs increased by a similar amount in both tissues of the old mice.

Dolichyl phosphate formed during the germination of isolated soybean embryos is derived primarily by the enzymatic phosphorylation of previously synthesized (stored) dolichol

Abstract The total dolichol and total dolichyl phosphate (Dol- P ) content of soybean seed embryos and cotyledons have been measured after saponification of the tissue. Non-germinated embryos and cotyledons each contain both dolichol and Dol- P . Dolichol intermediates from embryos have different homologue distribution patterns than those from cotyledons, although species containing 17 and 18 isoprene units predominate in both tissues. Although about 90% of both the total dolichol and total Dol- P of the whole seed is in the cotyledons, the concentration of both forms is about 4-fold higher in the embryo. In both tissues about 5% of the entire dolichol pool (total dolichol and total Dol- P combined) is phosphorylated. Dolichyl fatty acyl esters (Dol-FA) which account for 20% of the total dolichol in embryos are totally absent from the cotyledons. In embryos the fraction of the entire dolichol pool present in phosphorylated form increased from 5 to about 15% within 20 h of the start of germination, while the Dol-FA content did not change. The homologue distribution pattern of embryo dolichols also did not change during germination. Metabolic labeling studies with [3H]H2O and [32P]phosphoric acid, demonstrated that newly formed Dol- P is obtained primarily by the enzymatic phosphorylation of pre-existing (stored) dolichol.

Regulation of levels of dolichol and dolichyl phosphate during germination and early development of soybeans

In a previous study (Ravi, K., Rip, J.W. and Carroll, K.K. (1983) Biochem. J. 213, 513–518) soybeans were found to contain considerable amounts of dolichol but only traces of dolichyl phosphate (Dol-P). Further experiments have suggested that dolichol intermediates are involved in glycolipid and glycoprotein biosynthesis during germination, and have shown that the dolichol content of soybeans decreases over the first few days after germination, while the Dol-p content increases. These observations suggested the presence of an enzyme capable of phosphorylating preexisting endogenous dolichol (accompanying paper, Ravi, K., Rip, J.W. and Carroll, K.K. (1986) Biochim. Biophys. Acta 875, 618–625). Assays using soybean microsomes indicated that dolichol kinase activity increased over the first 3–4 days of germination and then became less active. After the third day, the dolichol content of seedlings increased gradually, whereas the concentration of Dol-p decreased until it was not detectable. The activity of Dol-P-phosphatase, a second enzyme involved in regulating Dol-p concentrations, increased over the first 5 days after germination. The phosphatase may have a biosynthetic role in supplying dolichol from de novo synthesis for (re)phosphorylation by dolichol kinase. All portions of 15-day-old seedlings: roots, stems, leaves and cotyledons contained dolichol, with the highest concentration in cotyledons. There appeared to be no difference in distribution of homologues among different parts of the plant or at different stages of development. Dol-p formed during germination may be utilized for synthesis of N-linked glycoproteins required during germination.

Extraction and quantitation of dolichol and dolichyl phosphate in soybean embryo tissue

A procedure for the quantitative extraction of both dolichol and dolichyl phosphate (Dol-P) in plant tissue (soybean embryos) into diethyl ether from an alkaline saponification mixture is described. A complete and quantitative separation of total dolichol and total Dol-P is then obtained based on their respective solubilities in diethyl ether and water. After separation dolichol and Dol-P can both be analyzed and quantitated directly by reverse-phase HPLC on C18 columns without additional purification. The two major homologs of dolichol and Dol-P are those with 17 and 18 isoprene units. The total dolichol and total Dol-P contents of dry embryos were 96.3 +/- 0.8 and 5.3 +/- 0.1 micrograms/g, respectively. The post-HPLC recoveries for dolichol and Dol-P were 101 +/- 2 and 84 +/- 3% respectively, using [1-14C]dolichol and Dol-P containing 20 isoprene units as recovery standards. Dol-P estimations could be carried out on material equivalent to as little as 65 mg embryo tissue.

Characterization of dolichol kinase from soybean microsomes

Microsomes from germinating soybeans contain a dolichol kinase with some unique and interesting properties. Kinase activity was linear with time, protein concentration and the concentration of one of the substrates, dolichol. The second substrate for all known dolichol kinases, CTP, was not required for maximal activity. CTP and other nucleoside tri- and diphosphates, particularly CDP, were actually inhibitory. In addition, 80% of maximal activity was observed in the absence of divalent cations, although a low level of stimulation was noted with Mg2+. The kinase reaction had pH optima at 7–8, and 9.5. Addition of nonlabeled dolichol to assays caused a proportional decrease in the incorporation of [l-14C]dolichol into [1-14C]dolichyl phosphate (Dol-p). Addition of nonradioactive Dol-p to incubations greatly increased [l-14C]Dol-p formation. Despite the unusual properties of the soybean enzyme, analysis by TLC and HPLC clearly indicated that Dol-p is the reaction product. The inhibitory and slight stimulatory effects of CDP and Mg2+, respectively, suggest that endogenous CTP and Mg2+ are preferentially utilized in the dolichol kinase reaction. These observations together with our recent study concerning developmental changes in concentrations of dolichol intermediates and changes in both dolichol kinase and dolichyl phosphate phosphatase activity (accompanying paper, Ravi K., Rip J.W. and Carroll K.K. (1986) Biochim. Biophys. Acta 875, 626–632) suggest that increased Dol-p synthesis is required for the formation of N-linked glycoproteins during germination.

Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing: Targeting Mobilized CD34+ Cells for Correction of Fabry Disease

Fabry disease is a rare lysosomal storage disorder (LSD). We designed multiple recombinant lentivirus vectors (LVs) and tested their ability to engineer expression of human α-galactosidase A (α-gal A) in transduced Fabry patient CD34+ hematopoietic cells. We further investigated the safety and efficacy of a clinically directed vector, LV/AGA, in both ex vivo cell culture studies and animal models. Fabry mice transplanted with LV/AGA-transduced hematopoietic cells demonstrated α-gal A activity increases and lipid reductions in multiple tissues at 6 months after transplantation. Next we found that LV/AGA-transduced Fabry patient CD34+ hematopoietic cells produced even higher levels of α-gal A activity than normal CD34+ hematopoietic cells. We successfully transduced Fabry patient CD34+ hematopoietic cells with “near-clinical grade” LV/AGA in small-scale cultures and then validated a clinically directed scale-up transduction process in a GMP-compliant cell processing facility. LV-transduced Fabry patient CD34+ hematopoietic cells were subsequently infused into NOD/SCID/Fabry (NSF) mice; α-gal A activity corrections and lipid reductions were observed in several tissues 12 weeks after the xenotransplantation. Additional toxicology studies employing NSF mice xenotransplanted with the therapeutic cell product demonstrated minimal untoward effects. These data supported our successful clinical trial application (CTA) to Health Canada and opening of a “first-in-the-world” gene therapy trial for Fabry disease.

Infantile Sialic Acid Storage Disease: Two Unrelated Inuit Cases Homozygous for a Common Novel SLC17A5 Mutation

Infantile sialic acid storage disease (ISSD) is a lysosomal storage disease characterized by accumulation of covalently unlinked (free) sialic acid in multiple tissues. ISSD and Salla disease (a predominantly neurological disorder) are allelic disorders caused by recessive mutations of a lysosomal anionic monosaccharide transporter, SLC17A5. While Salla disease is common in Finland due to a founder-effect mutation (p.Arg39Cys), ISSD is comparatively rare in all populations studied.Here, we describe the clinical and molecular features of two unrelated Canadian Inuit neonates with a virtually identical presentation of ISSD. Both individuals presented antenatally with fetal hydrops, dying shortly following delivery. Urinary free sialic acid excretion was markedly increased in the one case in which urine could be obtained for testing; postmortem examination showed a picture of widespread lysosomal storage in both. Both children were homozygous for a novel splice site mutation (NM_012434:c.526-2A>G) resulting in skipping of exon 4 and an ensuing frameshift. Analysis of a further 129 pan-Arctic Inuit controls demonstrated a heterozygous carrier rate of 1/129 (~0.4 %) in our sample. Interestingly, lysosomal enzyme studies showed an unexplained ninefold increase in neuraminidase activity, with lesser elevations in the activities of several other lysosomal enzymes. Our results raise the possibility of a common founder mutation presenting as hydrops in this population. Furthermore, if confirmed in subsequent cases, the marked induction of neuraminidase activity seen here may prove useful in the clinical diagnosis of ISSD.