Sequence and expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

Carrington, Mark; Bülow, Roland; Reinke, Heinz; Overath, Peter

doi:10.1016/0166-6851(89)90091-1

Cited by 60 publications

(45 citation statements)

References 26 publications

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“…PtdIns-glycan-specific phospholipase C from Trypanosoma brucei was also shown to be an amphiphilic protein (Hereld et al, 1988;Carrington et al, 1989); its amino acid sequence is 49% similar and 24% identical to the primary structure of PtdIns-glycan-specific phospholipase D from bovine liver as reported by Scallon et al (1991). Since the trypanosome enzyme was shown not to be associated with the plasma membrane but with membranes of intracellular organelles, Bulow et al (1989) suggested that it may be part of the endocytotic pathway.…”

Section: Discussionmentioning

confidence: 91%

Phosphatidylinositol‐glycan‐specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high‐density lipoproteins

Hoener

Brodbeck

1992

European Journal of Biochemistry

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Phosphatidylinositol (Ptd1ns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octylSepharose, concanavalin-A -Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 pgglml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycanspecific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.

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Section: Discussionmentioning

confidence: 91%

Phosphatidylinositol‐glycan‐specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high‐density lipoproteins

Hoener

Brodbeck

1992

European Journal of Biochemistry

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“…lacZ-CTA (italicized), and finally a sequence encoding the first 4 amino acids of GPI-PLCp (in lowercase lettering) (33,35). The reverse primer KCR5 contains a BamHI site (underlined), a stop codon (bold italicized), and the last 7 amino acids of GPI-PLCp (lowercase) (19,32). The amplification product was digested with BamHI and ligated into a BglII site of pUTK (34).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids-A GPI-phospholipase C plasmid pUTK-GPIPLC was obtained by insertion of a GPI-PLC coding region (19,32) and a translation enhancing 5Ј-untranslated region (33) into a Leishmania expression plasmid pXUTE-Kana R (pUTK) (34). The GPI-PLC insert was generated by PCR using a forward primer KCR4 (5Ј-TAAGGATCCTTAACACAGGAGGCAGCTAatgtttggtggtgta-3Ј) and the reverse primer KCR5 (5Ј-TATGTGGATCCTTAtgaccttgcggtttggt-3Ј).…”

Section: Methodsmentioning

confidence: 99%

“…GPI-PLCp is highly likely to bind to the cytoplasmic side of endosomes because it lacks an N-terminal ER signal peptide for entry into the secretory pathway (19,32). (Figs.…”

Section: Endosomes Glycosomes and Gpi Catabolism In L Majormentioning

confidence: 99%

“…(Lacking an N-terminal signal sequence (19,32), GPI-PLCp cannot enter the secretory system.) To test this hypothesis, Leishmania expressing GPI-PLCp were double labeled with antibodies against BiP, an ER marker (44) and GPI-PLCp, and observed by fluorescence microscopy.…”

Section: Endosomes Glycosomes and Gpi Catabolism In L Majormentioning

confidence: 99%

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Endosomes, Glycosomes, and Glycosylphosphatidylinositol Catabolism in Leishmania major

Zheng

Butler

Tweten

et al. 2004

Journal of Biological Chemistry

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Glycosylphosphatidylinositols (GPIs) serve as membrane anchors of polysaccharides and proteins in the protozoan parasite Leishmania major. Free GPIs that are not attached to macromolecules are present in L. major as intermediates of protein-GPI and polysaccharide-GPI synthesis or as terminal glycolipids. The importance of the intracellular location of GPIs in vivo for functions of the glycolipids is not appreciated. To examine the roles of intracellular free GPI pools for attachment to polypeptide, a GPI-specific phospholipase C (GPI-PLCp) from Trypanosoma brucei was used to probe trafficking of GPI pools inside L. major. The locations of GPIs were determined, and their catabolism by GPI-PLCp was analyzed with respect to the intracellular location of the enzyme. GPIs accumulated on the endo-lysosomal system, where GPI-PLCp was also de- tected. A peptide motif [CS][CS]-x(0,2)-G-x(1)-C-x(2,3)-Sx(3)-L formed part of an endosome targeting signal for GPI-PLCp. Mutations of the endosome targeting motif caused GPI-PLCp to associate with glycosomes (peroxisomes). Endosomal GPI-PLCp caused a deficiency of protein-GPI in L. major, whereas glycosomal GPI-PLCp failed to produce the GPI deficiency. We surmise that (i) endo-lysosomal GPIs are important for biogenesis of GPI-anchored proteins in L. major; (ii) sequestration of GPI-PLCp to glycosomes protects free protein-GPIs from cleavage by the phospholipase. In T. brucei, protein-GPIs are concentrated at the endoplasmic reticulum, separated from GPI-PLCp. These observations support a model in which glycosome sequestration of a catabolic GPI-PLCp preserves free protein-GPIs in vivo.

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Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol.

et al. 1995

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Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI‐specific phospholipase C (PI‐PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI‐PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8‐barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI‐PLC in complex with its inhibitor, myo‐inositol, at 2.6 A resolution (R factor = 19.5%). This substrate‐like inhibitor interacts with a number of residues highly conserved among prokaryotic PI‐PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI‐PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.

show abstract

Sequence and expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

Cited by 60 publications

References 26 publications

Phosphatidylinositol‐glycan‐specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high‐density lipoproteins

Phosphatidylinositol‐glycan‐specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high‐density lipoproteins

Endosomes, Glycosomes, and Glycosylphosphatidylinositol Catabolism in Leishmania major

Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol.

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