Biomedical Engineering Reference
In-Depth Information
ligand for the lectins calnexin and calreticulin (Brodsky and Skach
2011
) . The
membrane protein calnexin is associated with the translocon and binds substrates
co-translationally whereas calreticulin is soluble and interacts primarily with
secreted proteins after release from the ribosome.
Many G protein-coupled receptors are glycosylated on the N-terminus; likely,
G protein-coupled receptors are substrate to OST and therefore, glycosylation
occurs co-translationally. Immature as well as mature forms of receptors typically
appear as broad protein bands on Western blot reflecting the variable composition
of mature complex oligosaccharides. It has become common use to identify imma-
ture forms of the receptor on Western blot by the presence of a mannose-rich
core-glycosylated species the size of which can be specifically reduced by in vitro
incubation with endoglycosidase H. In the Golgi apparatus, receptors may in
addition be O-glycosylated on serine or threonine residues (Li et al.
2007
;
Hakalahti et al.
2010
) .
Experimental evidence has indicated that N-glycosylation may not be vital in
folding, maturation or ER export of G protein-coupled receptors (Hawtin et al.
2001
;
Li et al.
2007
), but this is not a general rule (Jayadev et al.
1999
) . Thus, among
G protein-coupled receptors the role of N-glycans is possibly non-redundant; how-
ever, there is a chance of ambiguous experimental results. The experimental approach
typically involves site-directed mutagenesis (to remove acceptor sites) or incubation
of cells with tunicamycin, an inhibitor of the transfer of UDP-N-acetylglucosamine
to dolichol phosphate. Disappearance of one acceptor site by mutagenesis opens up
the availability of others to N-acetyl glucosamin transferases and incubation with
tunicamycin may affect the complement of chaperones and transferases (in particular
of those that are glycosylated themselves) present in the ER. It is therefore possible
that some of the reported observations are due to compensatory adaptations rather
than an immediate consequence of an absent glycan. (Functional details of receptor
glycosylation are discussed elsewhere in this volume.)
1.2
Stability of the Receptor Structure
The efficiency of the folding process is dependent on the individual protein.
Polypeptides for which acquisition of the correct tertiary (and quaternary) structure
has failed usually are not transported through the secretory pathway. Rather,
they are dislocated into the cytosol and delivered to proteasomal degradation.
Controversial data have been published on the actual fraction of newly synthesized
chains that never attain native structure and are degraded. Values range up to 30%
or more and polytopic membrane proteins are among those that are most prone to
misfolding (Hebert and Molinari
2007
). Correct folding is examined by the
ER-bound quality control machinery, which can assess the conformation of protein
domains separately on the luminal and cytosolic side of the ER membrane; maybe
there is an additional and distinct mechanism to check the fold of the transmembrane
domain. Yeast has been employed as the model organism for assigning individual
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