D-Bifunctional protein

Acyl-CoA oxidase deficiency D-bifunctional protein deficiency Racemase deficiency Refsum s disease... 

Bifunctional protein deficiency. The enzyme defect involves the D-bifunctional protein. This enzyme contains two catalytic sites, one with enoyl-CoA hydratase activity, the other with 3-hydroxyacyl-CoA activity [13]. Defects may involve both catalytic sites or each separately. The severity of clinical manifestations varies from that of a very severe disorder that resembles Zellweger s syndrome clinically and pathologically, to somewhat milder forms. Table 41-6 shows that biochemical abnormalities involve straight chain, branched chain fatty acids and bile acids. Bifunctional deficiency is often misdiagnosed as Zellweger s syndrome. Approximately 15% of patients initially thought to have a PBD have D-bifunctional enzyme deficiency. Differential diagnosis is achieved by the biochemical studies listed in Table 41-7 and by mutation analysis. 

D-bifunctional protein deficiency [5], 2-methyl acyl-CoA racemase (AMACR) deficiency [3] and sterol carrier protein (SCP-x) deficiency [6], the disorders of etherphospholipid biosynthesis (dihydroxyacetone phosphate acyltransferase and alkyl- dihydroxyacetone phosphate synthase deficiency) [2], the disorders of phytanic acid alpha-oxidation (Refsum disease) [15], and the disorders of glyoxylate detoxification with hyperoxaluria type 1 as caused by alanine glyoxylate aminotransferase deficiency as a sole representative. 

Unfortunately, a minority of the patients with peroxisomal dysfunction cannot be diagnosed using plasma parameters. In the authors laboratory, patients have been seen with peroxisome biogenesis defects, D-bifunctional protein deficiency, and acyl-CoA oxidase deficiency in whom no abnormalities of plasma VLCFA, phytanic acid, pristanic acid or bile acids could be established. Hence, a strong clinical suspicion of peroxisomal disease should always be verified by fibroblast investigation, regardless of the outcome of plasma analyses. 

Su, H.M., Moser, A.B., Moser, H.W., and Watkins, P.A., Peroxisomal straight-chain Acyl-CoA oxidase and D-bifunctional protein are essential for the retroconversion step in docosahexaenoic acid synthesis, J. Biol. Chem., 276, 38115, 2001. 

Fig. 1. The revised pathway of 22 6n-3 biosynthesis from 18 3n-3. 18 3n-3 is converted to 24 6n-3 in microsomes then 24 6n-3 enters peroxisomes for one cycle of fatty acid (3-oxidation, via straight-chain acyl-CoA oxidase, D-bifunctional protein, and 3-oxo-acyl-CoA thiolase or sterol carrier protein X to produce 22 6n-3. The peroxisomal fatty acid (3-oxidation enzymes, branched-chain acyl-CoA oxidase, and L-bifunctional protein are not thought to participate in 22 6n-3 synthesis.

Fibroblast cell lines from patients with aldehyde oxidase 1 (AOxl) and D-bifunctional protein (DBF) deficiency accumulated metabolic intermediates between 18 3n-3 and 24 6n-3 similar to control cells when incubated with [l- C] 18 3n-3 (Table 2). However, the rate of 22 6n-3 synthesis was <10% of control in these cell lines, indicating that these 2 peroxisomal fatty acid P-oxidation enzymes are involved in the retrocon-version of 24 6n-3 to 22 6n-3. The involvement of AOxl in the synthesis of 22 6n-3 was also demonstrated in vivo by Infante et al. (21) who detected less radiolabeled 22 6n-3 synthesis in AOxl knockout mouse livers compared with control litter-mates after the intraperitoneal injection of [U- C]18 3n-3. 

Straight-chain acyl-CoA oxidase deficiency (AOxI) D-bifunctional protein (DBP)... 

D-BIFUNCTIONAL PROTEIN AND STEROL-CARRIER-PROTEIN X (SCPX) NEW INSIGHTS INTO THEIR FUNCTIONAL ROLE FROM STUDIES ON PATIENTS AND MUTANT MICE... 

Inde ndent studies from several laboratories also led to the identification of this enzyme. " In 1994 Novikov et cdJ had already found that peroxisomes contain multiple 3-hydroxyacyl-CoA dehydrogenases of which one turned out to be the newly recr -nized D-bifunctional protein. Systematic studies by the group of Hiltunen into the... 

However, until there is a unified nomenclature we will use the term D-bifunctional protein as suggested by Hashimoto next to L-bifunctional protein since the name... 

The second and third steps of peroxisomal P-oxidation are catalysed by two multifunctional enzymes D-bifunctional protein and L-bifunctional protein. Here we show that fibroblasts of a patient described as being deficient in the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein and fibroblasts of a patient described as being deficient in L-bifunctional protein do not complement one another. Using a newly developed method to measure the activity of D-bifunctional protein in fibroblast homogenates, we found that the activity of the D-bifunctional protein was completely deficient in the patient with presumed L-bifunctional protein deficiency. 

Until recently, P-oxidation of different fatty acids and fatty acid derivatives in peroxisomes was thought to be mediated by a single bifunctional protein containing both 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. " Recent studies have shown that there is an additional bifunctional protein alternatively called multifunctional protein 2 multifunctional enzyme 2, or D-bifunctional protein. " This newly identified bifunctional protein generates a D-3-hydroxyacyl-CoA, whereas the other bifunctional protein generates L-3-hydroxyacyl-CoA intermediates. Because of this remarkable difference in stereospecificity of the two bifunctional proteins, Hashimoto and coworkers suggested the names D-bifunctional protein (D-BP) and L-bifunctional protein (L-BP), respectively. ... 

We recently identified a new peroxisomal disorder in a patient showing signs and symptoms comparable to those observed in Zellweger syndrome. In the patients plasma, veiy-long-chain fatty acids, pristanic acid, and di- and trihydroxycholestanoic acid were elevated suggesting a defect in the peroxisomal P-oxidation system. Subsequent studies identified the defect in this patient at the level of the 3-hydroxyacyl-CoA dehydrogenase component of the newly identified D-bifunctional protein.""... 

Patient 1 suffered from an isolated deficiency of the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein and has been fully described elsewhere."" The clinical and biochemical characteristics of patient 2 have been described previously by Watkins and cowoikers."... 

The combined activity of the 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase components of D-bifunctional protein was measured as described in. °... 

Recently we described a patient with a defect in the 3-hydroxyacyl-CoA dehydrogenase component of the D-bifunctional protein. We have now performed complementation studies and fused fibroblasts from this patient (patient 1) with fibroblasts from the L-bifunctional protein defieient patient known from literature (patient 2) followed by measurement of pristanic acid P-oxidation in the fused cells (Table 1). As a control, cells were not fused but only cocultivated after which pristanic add P-oxidation was measured. Remarkably, pristanic acid P-oxidation was not restored after fusion, indicating that cells from patient 1 and patient 2 did not complement one anoflier. As expected, both cell lines did show complementation when fusions were performed with fibroblasts from a Zellweger patient (Table 1). 

The results of the complementation studies suggest that in both patient 1 and patient 2 the defect is in the same gene although patient 1 was describe as being D-bifimctional protein deficient and patient 2 was described as being L-bifunctional protein deficient. Importantly, both patients showed elevated levels of C26 0 and accumulation of bile acid intermediates (DHCA, THCA) in plasma " The accumulation of THCA in plasma and the deficient p-oxidation of pristanic acid in fibroblasts are hard to reconcile with the fact that L-bifunctional protein has been found not to be involved in the p-oxidation of 2-methyl branched-chain fatty acids, like pristanic acid, and bile acid intermediates, like THCA. In fact, recent studies have shown that it is the D-bifunctional protein which is involved in the P-oxidation of these substrates. This led us to measure the activity of D-bifunctional protein in fibroblasts from patient 2. 

To this end fibroblasts of patient 2 were incubated with the enoyl-CoA ester of THCA and formation of 24-hydroxy-THC-CoA and 24-keto-THC-CoA was measured (Fig. 1). The results in Fig.l elearly show that patient 1 is deficient at the level of the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein because of the increased formation of 24-hydroxy-THC-CoA and lack of 24-keto-THC-CoA formation (Fig. IB). In contrast, there was only very little formation of 24-hydroxy-THC-CoA and no formation of 24-keto-THC-CoA in patient 2 (Fig. 1C). 

These data strongly suggest that it is the D-bifimctional enzyme which is fimction-ally inactive in patient 2. Subsequent studies which include analysis at the molecular level, have revealed distinct mutations in the gene coding for D-bifunctional protein. These results will be described elsewhere (Van Grunsven et al., in preparation). 

Figure 1. D-Bifunctional protein activity measurement. Fibroblast homogenates were incubated with 24-ene-THC-CoA and the products 24-hydroxy-THC and 24-keto-THC-CoA were separated by HPLC. (A) control, (B) patient 1, (C) patient 2. Peaks are numbered as follows 1,24-hydroxy-THC 2,24-keto-THC-CoA 3, contaminant 4, (24 )-24-ene-THC-CoA.

Four defined disorders of peroxisomal jff-oxidation have been identified including (1) X-linked adrenoleukodystrophy/adrenomyeloneuropathy, (2) acyl-CoA oxidase deficiency (3) D-bifunctional protein deficiency and (4) peroxisomal thiolase deficiency. The clinical presentation of the latter 3 disorders resembles that of the disorders of peroxisome biogenesis whereas the clinical presentation of X-linked ALD/AMN patients is completely different which explains why it is better to discuss this entity separately under d. 

D-bifunctional protein deficiency (D-BP), the second peroxisomal -oxidation disorder, was only delineated recently [6-9]. All patients identified sofar (>40 see [10]) show severe clinical abnormalities including hypotonia, craniofacial dysmorphia, neonatal seizures, hepatomegaly, and developmental delay. Most patients with D-BP deficiency die in the first year of life. A remarkable observation is that patients with D-BP deficiency show disordered neuronal migration as in ZS. 

The situation with respect to the subsequent steps in peroxisomal j -oxi-dation has long remained an enigma but recent data have resolved this. It turns out that it is not the originally discovered bifunctional protein which forms and dehydrogenates L-3-hydroxyacyl-CoA esters but the more recently discovered D-bifunctional protein which forms and dehydrogenates D-3-hydroxyacyl-CoAs which is involved in the ) -oxidation of C26 0, pristanic acid and the two cholestanoic acids DHCA and THCA (Fig. 25.2). 

If a POD has been established, the nature of the true enzymatic defect needs to be determined using direct enzyme assays for acyl-CoA oxidase, D-bifunctional protein [8] and the peroxisomal thiolases [20] followed by molecular analyses. If no abnormalities are found, complementation analysis is warranted (see [10]). 

Peroxisomal fatty acid y9-oxidation deficiencies Acyl-CoA oxidase deficiency and D-bifunctional protein deficiency have both been resolved at the molecular level [10] and many often private mutations have been identified. The same is true for Refsum disease and 2-methylacyl-CoA racemase deficiency [16]. 

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