1.3.2.2.1 Influence of structural aspects of the LLO donor on OST reaction efficiency
Full-length lipid-linked core oligosaccharide (GlcsMangGlcNAci-PP-Dol) is the preferred glycosyl donor in vitro and in vivo in all eukaryotes (Trimble et al, 1980; Sharma et al, 1981; Runge et al, 1984; Verostek et a l, 1993). However, this is not the case in trypanosomatid protozoa, which lack the Dol-P-Glc synthase gene (Bosch et al,
1988) and hence produce only Man9GlcNAc2-PP-Dol as the LLO (Parodi, 2000a). The
presence of the three glucose residues on the LLO precursor in all other eukaryotes promotes more efficient transfer. The minimal structural requirement of the glycosyl donor in vitro is found to be GlcNAcz-PP-Dol, as no transfer could be demonstrated from GlcNAci-PP-Dol (Sharma et al, 1981). Alvarado and colleagues (1991) have postulated that the presence of glucose residues enhances transfer by providing a favourable conformation that contributes to oligosaccharide recognition by OST and by influencing the apparent binding affinity for the acceptor substrate (Sharma er al, 1981; Breuer and Bause, 1995; Knauer and Lehle, 1999). Reaction kinetics revealed that Km values for the peptide acceptor substrate of the yeast OST were altered by the structural composition of the LLO. The Km for the same acceptor peptide was found to be 10-
Chapter 1_____________________________________________________Introduction
G1cNAc2-Do1PP as the donor (Sharma et a l, 1981). The transfer of truncated oligosaccharides initially observed in vitro (Sharma et al, 1981) was also shown later to occur in vivo (Huffaker and Robbins, 1983). Studies using alg yeast mutants, with either a temperature sensitive block in LLO assembly or in alg null mutants that accumulate various oligosaccharides, provided the evidence that truncated oligosaccharide chains or fewer chains, depending on the protein, were being transferred onto the proteins, for example, Mani.2GlcNAc2 in alg2 (Jackson et a l, 1989,
1993).
The rate of transfer of truncated LLOs is not only inefficient by 20-30 fold, it requires a considerably longer time. As the rate of translation and translocation of nascent polypeptide is independent of co-translational modification such as ^-glycosylation (Jackson, 1989), inefficient transfer of truncated LLO results in the lack of occupancy of A^-glycosylation sites. Thus, the relative concentrations of normal and truncated LLOs could lead to non-glycosylation of a consensus sequon or its occupancy by an abnormal or truncated LLO. An altered pattern of glycosylation could affect the interaction of the glycoproteins with ER chaperones. Consequently this could have a profound effect on the folding of the protein and protein-protein interactions, which may determine its fate with respect to its intracellular degradation or exit from the ER to the Golgi apparatus for subsequent processing.
1.3.3 Processing of protein-bound oligosaccharides in the ER
Immediately after the transfer of Glc3Man9GlcNAc2-oligosaccharide to the Asn-residue
in the A^-glycosylation sequon, the terminal «-1,2-linked Glc residue is cleaved off by ER membrane-bound a-glucosidase I (Gls I). The Gls I is considered to be located in
Chapter 1_____________________________________________________Introduction
juxtaposition to OST. This enzyme is highly specific for the terminal a-l,2-linked Glc residue and its action converts the protein-linked oligosaccharide to Glc2Man9GlcNAc2-
structure. Using the ALG2 yeast strain, Jakob et al (1998) were unable to detect protein- bound Glc3Man9GlcNAc2. This reinforces the point that the removal of the terminal a-
1,2-linked Glc residue by Gls I is followed immediately by the OST transfer reaction. The Gls I can thus be postulated to function as a “guardian” of transfer of oligosaccharide to the polypeptide. OST, like any other transferase can act as a hydrolase immediately after the transfer and catalyse the reverse reaction as follows:
OST as transferase GlcsMançGlcNAci-PP-Dol + Asn-polypeptide ---►
OST as hydrolase GlcsMangGlcNAcz- Asn-polypeptide + Dohchol pyrophosphate + H2O
Asn-polypeptide + GlcgMan^GlcNAcz-OH + Dolichol pyrophosphate
The immediate removal of the terminal Glc residue by Gls I, prevents this undesirable action by OST as a reverse transferase or hydrolase.
The analysis of the structure of protein-bound oligosaccharides as well as their temporal appearance also showed that the removal of the first a-l,3-linked Glc by a-glucosidase II (Gls II) was a rapid process. Jakob et al (1998) further demonstrated that the mono- glucosylated GlciMan9GlcNAc2 was converted to Man9GIcNAc2 with a half-life of
approximately 2 minutes and this occurred before processing by mannosidase I (ERMI), which was a relatively slow process (half-life of 10 minutes) (Jakob et al., 1998). In the
Chapter I Introduction
the same kinetics in both Gls Il-deficient and Gls Il-proficient strains (Jakob et al.,
1998). p i,4 Pl,4 Dol-PP 2 1 Protein
ER
2 Protein 3 Protein 4 ProteinProtein Protein Protein Protein Protein Protein
Figure 1.7 Biosynthetic pathway of a bi-antennary N-glycan of a glycoprotein. The enzymatic steps involved are (1) OST transfers the precursor oligosaccharide from Dol-PP to nascent protein; (2) a-glucosidase I removes distal (al-2)-linked Glc, (3,4) a-glucosidase II removes distal (al-3)-linked Glc residues stepwise; (5) a-1,2- mannosidase I, Mang-mannosidase and a-mannosidase I remove the (al-2)-linked Man residues; (6) A^-acetylglucosaminyltransferase I adds the first GlcN Ac residue at a specific Man residue, (7) a-mannosidase II removes the two terminal Man residues, (8) A-acetylglucosaminyltransferase II adds a GlcN Ac residue to newly generated Man residue, (9) (5-1,4-galactosyltransferase adds a Gal residue on each GlcN Ac; (10) a-2,3- sialyltransferase terminates the glycan with an A-acetylneuraminic acid (NeuSAc) on each Gal residue. ■ = GlcNAc,o = Man, O = Glc, o = Gal, A = Neu5 Ac, A = ftjcose (may also be added during subsequent terminal glycosylation).
The protein-bound oligosaccharide undergoes further processing in the ER, which involves the removal of some o f the Man residues. A number of a-mannosidases have been identified, localized specifically in the ER or Golgi and cytoplasm (Daniel et a i,
Chapter 1 Introduction
ERMI and ERMII, of which ERMI is more abundant (Moreman et al, 1994). They both produce MangGlcNAcz isomers as shown in Figure 1.8.
ERMI M s M y Mg I I I M4 M e Mg Mz ' ^ u / M / I Gnz I Gni I Asn ERMII M s Mg I I M4 M e Mg I M2
\ /
M/ I Ghz I Gni I Asn Man 8B isomer M s My I I M 4 M e M g Mz Ml I Gnz I Gni I Asn Man 8C isomerFigure 1.8 Mannose processing in the ER and the production of MangGlcNAci isomers.
Another ER a-mannosidase, described as Mang-mannosidase, which processes Man9GlcNAc2 to MangGlcNAcz (Fig 1.9) has been reported by Bause et al, (1992).
Chapter 1 Introduction Ms M7 M9 I M4 1 Mé 1 Ms 1 Mz