Glycosylation of thrombospondin type 1 repeats

In the course of mutagenesis of the forth thrombospondin type 1 repeat (TSR4) of the
axonal guidance protein F-spondin, one mutant designated ΔDS2,3-TSR4 missing two
out of three cystein bridges was found to lack the disaccharide Glcβ1,3-Fuc-O. This
protein was demonstrated to be secreted as efficiently as wild type TSR4. In gel filtration
experiments unusual features of folding could not be observed. Analysis of the protein
by mass spectrometry indicated that the module had acquired an additional
glycosylation. Using a combination of tandem CID MS and digestion with glycosidases,
it was demonstrated that the tetrasaccharide corresponds to a biantennary
NeuNAcα2,3Galβ1,3[NeuNAcα2,6]GalNAc-O- glycan. This carbohydrate structure is
also known as the disialyl-T-antigen. The glycan could be demonstrated to be attached
exclusively to the same residue (Thr-601) that carries the disaccharide Glcβ1,3-Fuc-O in
wild type TSR4. ΔDS2,3-TSR4 is consequently an experimental proof that one residue
can be modified with two different types of glycosylation. Since the disaccharide
Glcβ1,3-Fuc-O is transferred in the Endoplasmatic Reticulum and the disialyl-T-antigen
is added in the Golgi, ΔDS2,3-TSR4 has undergone a glycosylation shift involving
different cellular organelles. In order to identify the enzymes that recognized the module
and modify the Thr-601 residue ΔDS2,3-TSR4-derived peptides were screened in an in
vitro assay. It was found that GalNAc T1 and GalNAc T3 are capable to add GalNAc
moieties. GalNAc T1 was found to glycosylate as well Thr-601 and Ser-596. GalNAc T3
was identified to be specific for Thr-601, thus mimicking the situation observed in vivo.
The role of this transferase could be further confirmed in vivo by co-expression
experiments of GalNAc T3 together with ΔDS2,3-TSR4 in CHO-K1 cells. These
experiments resulted in significant increase of the disialyl-T-antigen on glycopeptides of
ΔDS2,3-TSR4. The results further demonstrated that after initiation of mucin-type Oglycosylation
by a glycosyltransferase such as GalNAc T3 the carbohydrate is extended
to the tetrasaccharide of a disialyl-T-antigen type in various cell types. Interestingly the
same O-glycan was also identified in the rat axonal guidance protein F-spondin. Fspondin
had previously been described to be C-mannosylated on tryptophans and to
contain the disaccharide Glcβ1,3Fuc-O-Ser/Thr. Using low energy CID tandem MS in
combination with glycosidases and specific sugar-binding lectins, it was possible to map
the disialyl-T-antigen to a peptide located in the N-terminal reelin/spondin domain. Fspondin
is therefore the first protein known to contain C-mannosylation, the disaccharide
Glcβ1,3Fuc-O-Ser/Thr, N-glycosylation and also the tetrasacchride NeuNAcα2,3Galβ1,3
[NeuNAcα2,6]GalNAc-O-.
In order to study the glycosylation of thrombospondin type 1 repeats of F-spondin in
more detail, the fourth (TSR4) and four consecutive thrombospondin type 1 repeats
(TSR1-4) were expressed in the mammalian cell line HEK-EBNA. In order to facilitate
purification, the proteins were expressed as fusion proteins designated TSR4fchis and
TSR1-4fchis. The proteins were purified in high amounts and C-mannosylation as well
as the modification with the disaccharide Glcβ1,3-Fuc-O were examined by LC-MS. It
was found that the TSR4fchis and TSR1-4fchis proteins carried C-mannosylation on
their tryptophans and Glcβ1,3-Fuc-O on their serines or threonines in the consensus
sequence C1X2–3S/TC2X2G. Quantification of the amount of glycosylation on the
TSR4fchis protein revealed that the first tryptophan in the WXXW motif is almost
quantitatively C-mannosylated, the second tryptophan shows partial C-mannosylation
and the threonine in the consensus sequence C1X2–3S/TC2X2G was predominantly
modified with the disaccharide Glcβ1,3-Fuc-O. In order to have non-glycosylated TSRs,
TSR4fchis and TSR1-4fchis were expressed in the methylotrophic yeast Pichia pastoris.
TSR4fchis could be successfully expressed and purified in amounts comparable to
correctly folded TSR4 expressed in E. coli. It was found that this host is negative for Cmannosylation
and O-fucosylation. However, indirect evidence indicated, that TSR4fchis
expressed in yeast had undergone hyperglycosylation of the fusion protein. Therefore a
direct comparison of TSR4fchis from HEK-EBNA and TSR4fchis from P. pastoris was
not possible.