Microheterogeneity of the Hydrophobic and Hydrophilic Part of the Glycosylphosphatidylinositol Anchor of Alkaline Phosphatase from Calf Intestine

Abstract

Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl-(Etn-P),-glycosyl-PtdIns with HF generated 8 glycosyl-Ins species the largest of which (G1 and G2) have the following proposed structures:G1 aManand G5 are lower homologues of G1 and G2, respectively, being one a l -2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N-acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic Man,Glc-Ins species are either substituted with an N-acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular-mass determination by matrix-assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO,. Purification of the glycosylinositol species was achieved by a novel reverse-phase HPLC technique using fluorescent fluoren-9-yl-methoxycarbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level.We observed recently that native alkaline phosphatase separates on octyl-Sepharose into four distinct fractions of increasing hydrophobicity (F1 -F4). Here we show that all four fractions contain G1 -G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residueshbunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10 % which is in contrast to most glycosyl-PtdIns-anchored proteins of mammalian origin.

Specific inhibition of GPI-anchored protein function by homing and self-association of specific GPI anchors

“…This may help determine signaling specificity, as a close and specific association occurs between some gangliosides and certain signaling molecules (Kasahara et al, 1997; Iwabuchi et al, 1998). Similarly, GPI anchors are heterogeneous, as differences in both hydrophobic and hydrophilic GPI anchor regions have been documented (Ferguson et al, 1988; Armesto et al, 1996). These raft and anchor variations may be sufficient to create GPI anchor–specific membrane domains with different protein repertoires.…”

Formation and Remodeling of Inositolphosphoceramide during Differentiation of Trypanosoma cruzi from Trypomastigote to Amastigote

“…6A, lanes 2 to 6) and trypomastigotes (Fig. 6B, lanes 2 to 6) for different times (5,15,60, and 120 min and 18 h). Also, the activity in the supernatant of the membrane preparation was analyzed in an overnight incubation (Fig.…”

“…The substrate dissolved in chloroform-methanol (1:1, vol/vol) was dried in the incubation tubes under a N 2 stream, and the membrane fraction, suspended in 100 l of buffer, was added. After incubation in a shaker at 30°C for different times (5,15,60, and 120 min and 18 h), the reaction was stopped by addition of 660 l of chloroformmethanol (1:1, vol/vol). A control incubation of the substrate without membranes was also done.…”

“…The inositolphospholipids (IPLs) of these anchors can possess either a glycerolipid or a ceramide. In contrast, only acylglycerophospholipids have been identified in the anchor of T. brucei glycoproteins (17,18), while diacyl-or alkylacylglycerolipids are part of the anchors of mammalian glycoproteins (5,25,30). Several important glycoproteins of T. cruzi are attached to the cell surface via a ceramide-containing anchor linked to the C terminus of the protein (24), such as the Ssp4 antigen of amastigote forms (3,7), the unique trans-sialidase glycoprotein (2), and the mucins of metacyclic trypomastigotes (1).…”

Insulin-Mimicking Bioactivities of Acylated Inositol Glycans in Several Mouse Models of Diabetes with or without Obesity

“…In contrast, less bioactive IG-15 with a 1-palmitoyl-group and alpha 1,3 linkage might not to be generated from GPI precursor. It has been reported that a minor fatty acid constituent found at the 2-position of myo-inositol in mammalian GPI is myristate [52]. We synthesized IG-21 (D-glucosamine-(α1,6)-(2-myristoyl)-D-myo-inositol) and other short-chain fatty acid (C2 to C12)-linked inositol glycans species and checked insulin-mimic bioactivities in 3T3-L1 adipocytes.…”