Review: An evolving view of the eukaryotic oligosaccharyltransferase

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Review An evolving view of the eukaryotic
oligosaccharyltransferase

• Presented by Yin Wu

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Introduction

• Asparagine-linked glycosylation (ALG) is one of
  he most common protein modification reactions in
  eukaryotic cell.
• This review introduces
• The structure and function of the eukaryotic
  oligosaccharyltransferase (OST)
• Genetic homologues analysis of the
  archeabacterial and eubacterial OST.

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Content

• Overview of the process of glycosylation
• Oligosaccharide donors for the OST
• Acceptor substrates and the Donor-substrate
  activation model
• Sequence context factors that influence
  N-glycosylation
• OST subunit composition and gene homologues
  analysis of OST.

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Overview of the Process of Glycosylation

• N-linked glycosylation occurs on the asparagine
  site of the protein. The glycosylated asparagines
  are in an N-X-T/S motif.
• The process of glycosylation (in the scope of
  this paper)
• The oligosaccharide donor is assembled on the
  carrier by sequential addition of monosaccharides
  with the help of glycosyltransferase (ALG
  proteins)
• The fully assembled ogligosaccharide is
  transfered to the specific N site on the
  polypeptide, which is then synthesized into
  protein.

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Oligosaccharide Donors for the OST

• The oligosaccharide consists of three categories
  of monosaccharides namely GlcNAc, Man and Glc.
• Different monosaccharide residues are transferred
  by different glycosyltransderases. (Fig. 1)
• Defects in donor assembly are responsible for the
  disease known as congenital disorders of
  glycosylation (CDG-I).

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Sequence Context Factors That Influence
N-glycosylation

• N-glycosylated asparagine residues are within an
  N-X-T/S/C consensus sequon, where X can be any
  residue except proline.
• N-X-T servers as a better substrates than N-X-S,
  which is in turn much better than N-X-C.
• Overlapped sequons result in a single
  glycosylation site. E.g. N-N-T/S-T/S

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Sequence Context Factors That Influence
N-glycosylation (contd)

• Sequons inserted into short lumenal loops are not
  glycosylated.
• Glycosylation sites near the C-terminus of
  proteins are also less likely to be utilized.
• Mutations that prevent folding of a glycoprotein
  may trigger glycosylations at sites which are
  normally not glycosylated.

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OST Subunit Composition

• 8 polypeptides are identified in the yeast OST
  complex, namely Ost1p, Ost2p, Ost3p or Ost6p,
  Ost4p, Ost5p, Wbp1p, Swp1p and Stt3p.
• The interactions between the subunits are
  unclear.
• The Stt3 subunit contains the catalytic site of
  the OST.

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Acceptor substrates and the Donor-substrate
activation model

• Fully assembled donor is transferred 5-20 times
  faster than an assembly intermediate.
• A donor substrate activation model is proposed to
  explain the donor-substrate selection. (Fig 2)

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Gene Homologues Analysis of OST Subunits

• The Stt3 subunit is the most highly conserved
  polypeptide in the OST complex.
• Phylogenetic tree analysis shows that Stt3B
  proteins are more closely related to the fungal
  and nematode Stt3 proteins, whereas the Stt3A
  protein cluster includes Stt3 from D. discoideum.
  (?)

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Fig 3 Pylogenetic tree analysis of STT3
proteins. Representative fungal and metazoan Stt3
sequences as well as all currently available
protists sequences were selected for alignment.
Several archaebacterial and eubacterial Stt3
homologues were used to root the phylogenetic
tree.
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Gene Homologues Analysis of OST Subunits

• The Ost4 sequences are aligned between different
  species.
• Replacement of hydrophobic residues in the TM
  span of Ost4 with lysine or aspartic acid caused
  temperature sensitive growth and destabilized the
  interactions between the Ost4 and other subunits.

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Fig 4 Alignment of eukaryotic Ost4 sequences.
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Conculsion
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Discussion