posted on 2011-09-06, 00:00authored byMarcie
B. Jaffee, Barbara Imperiali
The central enzyme in N-linked glycosylation is the oligosaccharyl
transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked
carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology
to the Stt3 subunit of the complex multimeric yeast OTase. Sequence
identity between PglB and Stt3 is low (17.9%); however, both have
a similar predicted architecture and contain the conserved WWDxG motif.
To investigate the relationship between PglB and other Stt3 proteins,
sequence analysis was performed using 28 homologues from evolutionarily
distant organisms. Since detection of small conserved motifs within
large membrane-associated proteins is complicated by divergent sequences
surrounding the motifs, we developed a program to parse sequences
according to predicted topology and then analyze topologically related
regions. This approach identified three conserved motifs that served
as the basis for subsequent mutagenesis and functional studies. This
work reveals that several inter-transmembrane loop regions of PglB/Stt3
contain strictly conserved motifs that are essential for PglB function.
The recent publication of a 3.4 Å resolution structure of full-length C. lari OTase provides clear structural evidence that these
loops play a fundamental role in catalysis [Lizak, C.; (2011) Nature 474, 350−355]. The current study provides biochemical support for the role of
the inter-transmembrane domain loops in OTase catalysis and demonstrates
the utility of combining topology prediction and sequence analysis
for exposing buried pockets of homology in large membrane proteins.
The described approach allowed detection of the catalytic motifs prior
to availability of structural data and reveals additional catalytically
relevant residues that are not predicted by structural data alone.