Characterization of a cDNA encoding rat sterol carrier protein2

Sterol carrier protein2 (SCP2) is a 13.2-kD protein that is thought to be involved in the intracellular transport of cholesterol. Using synthetic oligonucleotides based on the protein sequence of SCP2, a clone (SP43) was isolated from a rat liver cDNA library. The DNA sequence revealed that the cDNA could encode a polypeptide of 273 amino acids (28.9 kD) or 143 amino acids (15.3 kD) in which the carboxy-terminal 123 amino acids are identical to the SCP2 protein. RNA blot hybridization revealed that a variety of rat tissues contain a homologous RNA of a size similar to SP43 (approximately 1.5 kb). Levels of SCP2 mRNA increased in parallel with cytochrome P450scc mRNA in the immature gonadotropin-primed rat ovary. The isolation of a cDNA clone encoding SCP2 will facilitate studies on its role in cholesterol metabolism.     

New aspects of sterol carrier protein 2 (Nonspecific lipid-transfer protein) in fusion proteins and in peroxisomes

Sterol carrier protein 2 (SCP2) is a 13-kDa peroxisomal protein, identical to nonspecific lipidtransfer protein, and stimulates various steps of cholesterol metabolism in vitro. Although the name is reminiscent of acyl carrier protein, which is involved in fatty acid synthesis, SCP2 does not bind to lipids specifically or stoichiometrically. This protein is expressed either as a small precursor or as a large fusion (termed SCPx) that carries at its C-terminal the complete sequence of SCP2. SCPx exhibits 3-oxoacyl-CoA thiolase activity, as well as sterol-carrier and lipid-transfer activities. The N- and C-terminal parts of SCPx are similar to the nematode protein P-44 and the yeast protein PXP-18, respectively. P-44, which has no SCP2 sequence, thiolytically cleaved the side chain of bile acid intermediate at a rate comparable to that of SCPx. This, together with the properties of other fusions with SCP2-like sequence, suggests that the SCP2 part of SCPx does not play a direct role in thiolase reaction. PXP-18, located predominantly inside peroxisomes, is similar to SCP2 in primary structure and lipid-transfer activity, and protects peroxisomal acyl-CoA oxidase from thermal denaturation. PXP-18 dimerized at a high temperature, formed an equimolar complex with the oxidase subunit, and released the active enzyme from the complex when the temperature went down. This article attempts to gain insight into the role of SPC2, and to present a model in which PXP-18, a member of the SCP2 family, functions as a molecular chaperone in peroxisomes.     

cDNAs encoding members of a family of proteins related to human sterol carrier protein 2 and assignment of the gene to human chromosome 1 p21----pter.

Sterol carrier protein 2 (SCP2) is believed to play a key role in intracellular lipid movement. Here we report the cloning and nucleotide sequences of cDNAs encoding SCP2-related proteins of 58.85 kD and 30.8 kD and the assignment of the SCP2 gene to human chromosome 1 p21-pter. The SCP2-related proteins share common deduced carboxyl amino acid sequences with SCP2 and the cDNAs have a common 3' untranslated nucleotide sequence. The mRNAs encoding these proteins increased in a coordinate fashion as human placental cytotrophoblasts differentiated into syncytiotrophoblasts in culture. Our observations document the existence of a family of related proteins encoded by the human SCP2 gene.     

Identification of SCP2165, a new SCP2-derived plasmid of Streptomyces coelicolor A3(2)

AIMS: Characterization of SCP2165, a plasmid identified in the Gram-positive bacterium Streptomyces coelicolor A3(2). METHODS AND RESULTS: Pulsed-field gel electrophoresis (PFGE) of mycelia of a S. coelicolor strain embedded in low melting agarose revealed the presence of a plasmid. Restriction enzyme mapping and sequence analysis of a 2.1 kb fragment revealed that this plasmid could be SCP2. SCP2 and its spontaneous derivative SCP2* are self-transmissible plasmids and have chromosome mobilizing ability (c.m.a.). SCP2* has a c. 1000-fold increased c.m.a. compared with SCP2. Interestingly the plasmid, named SCP2165, shows a c.m.a. from 5x10(-2) to 1x10(-1) which is 50-100-fold higher than that described for crosses involving SCP2*. CONCLUSIONS: SCP2165 is a SCP2 derivative plasmid with the highest c.m.a. so far described for SCP2 derivative plasmids. PFGE, under conditions we used, seems to be a fast way to identify large circular plasmids in Streptomyces strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Further knowledge of the SCP2 family may allow the construction of improved SCP2-derived cloning vectors. SCP2165 could be a potential tool for conjugational transfer of gene clusters between different Streptomyces species.     

Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein,in Intracellular Cholesterol Trafficking in Testicular Leydig Cells

Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking.     

SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat.

In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.     

Amino acid sequence of alpha chain of sarcoplasmic calcium binding protein obtained from shrimp tail muscle

The isotypes of sarcoplasmic Ca2+ binding protein (SCP) were purified from shrimp tail muscle. SCP exists in a dimeric form. One sample of shrimp contained only alpha A chain, whereas another contained alpha B and beta chains, and a heterodimer of alpha B beta which was not analyzed precisely. The amino acid sequences of the two alpha chains were determined. The two alpha chains are composed of 190 and 192 amino acid residues, respectively. The sequences of the two alpha chains differed in only four amino acids out of 192 residues. The sequences indicate that the alpha chain has three Ca2+-binding sites which are common to EF-hand type Ca2+-binding protein. In the absence of added Ca2+ and Mg2+, the amounts of bound Ca2+ in alpha A, alpha B, and beta chains were 3.0, 3.3, and 2.4 mol/22,000 g protein, respectively. Thus, it is suggested that all three isotypes of shrimp SCP have three Ca2+-binding sites which have high affinity to Ca2+. The sequence homology of shrimp SCP with other EF-hand type Ca2+-binding proteins is very low. The protein having the greatest homology with this SCP was cod parvalbumin; the sequence homology is 18%.     

Purification, characterization and comparison with mammalian SCP2 of a chicken SCP2-like protein.

1. Three proteins have been isolated from chicken (Gallus domesticus) liver that bind antibodies directed against authentic rat sterol carrier protein2 (SCP2) and have similar molecular mass to the three major immunoreactive rat liver proteins (12 kDa, 30-36 kDa, 55-60 kDa). 2. Bile from both chicken and rat contains the high molecular mass immunoreactive species. 3. The chicken 12 kDa SCP2-like protein purifies similarly to rat SCP2 but the homogeneous chicken SCP2-like protein is dissimilar in amino acid composition and N-terminal amino acid sequence. 4. The activity of chicken SCP2-like protein differs from rat SCP2 in that it was consistent with fusion (transfer of both polar surface and non-polar core lipids) rather than transfer of polar lipids only.     

Primary sequence and structural analysis of sterol carrier protein 2 from rat liver: homology with immunoglobulins

Sterol carrier protein 2 (SCP2) is involved in the later steps of cholesterol biosynthesis and in the intracellular transport of cholesterol. In the present investigation, the amino acid sequence of SCP2 from rat liver has been determined. It is a single polypeptide chain with 122 amino acid residues. Secondary structure prediction indicates an amphipathic alpha-helix region for residues 21-34 and antiparallel beta-sheet structure for residues 35-95. A major finding is the significant homology which exists over approximately 80 residues between SCP2 and the variable domains of the heavy chain of immunoglobulin G.     

Translational control of the circadian rhythm of liver sterol carrier protein. Analysis of mRNA sequences with a specific cDNA probe

The striking changes in amount of rat liver SCP (sterol carrier protein) during a 24-h dark-light cycle are due to alterations in the relative synthetic rate of SCP. However, functional SCP mRNA, measured by a cell-free translational assay, does not fluctuate in the dark-light cycle. Since cell-free translational assays do not always reflect the actual abundance of an mRNA molecule, a specific cDNA hybridization assay was used to directly quantitate SCP mRNA sequences. The cDNA probe was selected from a rat liver library by hybridization to a mixture of synthetic oligonucleotides containing a portion of the sequence of SCP mRNA. The relative amount and size distribution of the SCP mRNA species (approximately 700-800 nucleotides) does not change during the diurnal cycle. To explore possible mechanisms of this translational control, the polysomal distribution of SCP mRNA was compared at the maximum and minimum points of SCP synthesis. No significant amounts of SCP mRNA were present in nonpolysomal ribonucleoprotein particles. Furthermore, no alteration in the relative level of SCP mRNA associated with polysomes or in polysome size occurs at the maximum and minimum points of SCP synthesis. Thus, changes in total SCP mRNA levels or its polysomal distribution cannot account for the diurnal variation in SCP synthesis.     

Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to “buffer” cytosolic Ca²⁺. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3′ terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6–81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca²⁺ is linked.     

Integrated structures of the linear plasmid SCP1 in two bidirectional donor strains of Streptomyces coelicolor A3(2)

The linear plasmid SCP1 is integrated into the central region of the chromosome of Streptomyces coelicolor A3(2). The integrated structures of SCP1 in two bidirectional donor strains, 2612 and A634, were analyzed by cloning and sequencing of the junctions between the SCP1 DNA and the chromosomal DNA. In the NF (normal fertility) strain 2612, SCP1 is integrated in a right-handed direction into ORF-X at the left end of the IS cluster in AseI fragment E. An almost intact left end of SCP1 is retained, while the right terminal inverted repeat (TIR-R) of SCP1 and a 33-kb chromosomal DNA segment including the IS cluster are deleted. In the NF-like strain A634, SCPI is also integrated into AseI fragment E in a left-handed direction. The left junction is composed of IS466 with complete deletion of TIR-R of SCP1, and the right junction is located at the left end of IS468A* with half of TIR-L being deleted. During the integration event, a 5.4-kb chromosomal DNA segment including IS468A, IS468B, IS469 and IS466A was duplicated so that this sequence is now present on both sides of SCP1. Since 2612 and A634 exhibit a similar bidirectional gradient of gene transfer, it is surprising that their chromosomal structures are so different.     

Conformational plasticity of the lipid transfer protein SCP2

The nonspecific lipid transfer protein sterol carrier protein 2 (SCP2) is involved in organellar fatty acid metabolism. A hydrophobic cavity in the structure of SCP2 accommodates a wide variety of apolar ligands such as cholesterol derivatives or fatty acyl-coenzyme A (CoA) conjugates. The properties of this nonspecific lipid binding pocket are explored using NMR chemical shift perturbations, paramagnetic relaxation enhancement, amide hydrogen exchange, and 15N relaxation measurements. A common binding cavity shared by different physiological ligands is identified. NMR relaxation measurements reveal that residues in the three C-terminal alpha-helices within the lipid binding region exhibit mobility at fast (picosecond to nanosecond) and slow (microsecond to millisecond) time scales. Ligand binding is associated with a considerable loss of peptide backbone mobility. The observed conformational dynamics in SCP2 may play a role for the access of hydrophobic ligands to an occluded binding pocket. The C-terminal peroxisomal targeting signal of SCP2 is specifically recognized by the Pex5p receptor protein, which conducts cargo proteins toward the peroxisomal organelle. Neither the C-terminal targeting signal nor the N-terminal precursor sequence interferes with lipid binding by SCP2. The alpha-helices involved in lipid binding also mediate a secondary interaction interface with the Pex5p receptor. Silencing of conformational dynamics of the peptide backbone in these helices upon either lipid or Pex5p binding might communicate the loading state of the cargo protein to the targeting receptor.     

Nucleotide sequence analysis of the unusually long terminal inverted repeats of a giant linear plasmid, SCP1.

SCP1 is a giant linear plasmid of 350 kb coding for the methylenomycin biosynthetic genes in Streptomyces coelicolor. The unusually long terminal inverted repeats present on both ends of SCP1 were analyzed on the nucleotide sequence level. Analysis of six clones containing the terminal 0.35-kb XbaI fragment revealed a slight heterogeneity in the nucleotide sequences of the SCP1 ends. Moreover, it was indicated that this fragment contained seven palindromic inverted repeats and a GT-rich region in the 5'-end strand. The size of the terminal inverted repeats was determined to be 81 kb by the cloning and sequencing of their end-points. An insertion sequence, IS466 was shown to be present just at the end of the right terminal inverted repeat.     

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces

Non-transmissible derivatives of the Streptomyces multi-copy plasmid pIJ101 were mobilized, by co-integrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coeli-color A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.