Download Discussion

5 Discussion
5 DISCUSSION
The main subjects in this thesis were to study the secretion of a recombinant serglycin in the
MDCK II cell line and in an endothelial cell line. To enable easy detection and isolation of the
serglycin molecule, a C-terminal His-flag was added to the protein core. After cloning of this
recombinant serglycin-His-flag, both the MDCK II cell line and two different endothelial cell
lines (HUVEC and HAEC), were chosen for transfection experiments. Due to the non-successful
transfection of the endothelial cells, the project was reduced to study the secretion of serglycin in
the MDCK II cell line only. The use of the recombinant serglycin-His-flag required both
characterisation of the transfected MDCK II cell clones, at both mRNA and proteoglycan
secretion levels, and an optimisation of a Ni2+ Hi-Trap chelating chromatography system for
isolation of the recombinant serglycin-His-flag.
5.1 Expression of serglycin-His-flag in endothelial cells
5.1.1 Transfection of endothelial cells
It was not possible to obtain stably transfected endothelial cells. Both a HUVEC cell line and a
HAEC cell line were transfected, with several transfection methods. The HUVEC cell line was
more difficult to transfect than the HAEC cell line, but it was more tolerant against the different
transfection reagents. For instance, it tolerated the calcium phosphate method, which killed
almost all HEAC cells. Another lipid transfection reagent, lipofectamine, was also tested, but it
was found to be more toxic to the HUVEC cell line than the lipofectin reagent (result not
shown). Lipofectamine was not tested on HAEC cells.
The initial experiments revealed that the HAEC cell line was easier to transfect than the HUVEC
cell line. Thus, since it seemed easier to get stably transfected HAEC cells, only this cell line was
chosen for the further transfection experiments. In the optimised experiment, approximately 10
percent of the HAEC cells were transiently transfected with the lipofectin reagent, which should
be enough for obtaining stable transfectants. In this, and in the repeated experiments, some
transfected cells even survived considerably longer than control cells, which indicated that they
were stably transfected. However, it was not possible to expand these single cell colonies, and
stably transfected subclones were not obtained.
There are several possibilities why the endothelial cells does not grow after selection. A common
observation was that the surviving cells often were alone, without contact with other
neighbouring cells. This cell to cell contact may be important for the endothelial cells. During
culturing, the endothelia secretes various products, which stimulate growth of neighbouring cells.
With only a few living cells in the well, the concentration of these products may be too low, and
thus the cells die, due to lack of growth stimulation and cell communication. This is supported by
the observation that, when the wild type HUVEC cells became too diluted after splitting, they
often fail to divide, or divided at a very low rate. Interestingly, the transfected cells also often
died the day after exchange of the culture medium, which further indicates that this may be a
relevant factor. A way to overcome this might be to culture the cells in conditioned medium, or
maybe, not to exchange the culturing medium at all, before the cells expand to moderate cell
colonies. However, due to the extensive work on characterisation and isolation of serglycin-Hisflag secreted by the transfected MDCK II subclones, we decided to give priority to that work.
129
5 Discussion
Although it was not possible to stably transfect an endothelial cell line, the transfected MDCK II
cells have given important information about the GAG attachment on the serglycin core protein.
The proteoglycan synthesis in primary HUVEC cells has been investigated previously (Bergli,
1997). Of the total 35(S)macromolecules secreted by primary HUVEC cells, 30-40 % is CSPG
and 30-40 % is HSPG, equally secreted to both the apical and the basolateral compartments.
Since these endothelial cells also synthesise HSPG, it is possible that the serglycin core protein
can be modified with both CS-GAG and HS-GAG in HUVEC cells, as found for the transfected
MDCK II cells. Whether this attachment is regulated, and if the different variants are secreted to
different compartments, would be interesting to find out.
5.2 Expression of serglycin-His-flag in MDCK II cells
5.2.1 Characterisation of transfected MDCK II cells
The MDCK II cell line was successfully stably transfected with the recombinant serglycin-Hisfalg, and several different subclones were obtained. The mRNA analysis revealed that the
different transfected subclones varied in serglycin-His-flag mRNA expression (figure 4-11.B).
Some of the cells did not express the recombinant serglycin-His-flag molecule, even though they
survived the selective marker. It is not determined if these subclones have introduced the
pcDNA3.1(serglycin)/Myc-His vector into the genome, but it is unlikely that they could survive
the selection pressure marker without expressing the neomycin resistance gene. Most likely the
DNA sequence required for expression of the serglycin-His-flag has been ejected, or divided
during the insertion of the pcDNA3.1(serglycin)/Myc-His vector. An insertion of the vector
without serglycin mRNA expression is proven for subclone 1-6, since this subclone has altered
phenotype. It resembles more a muscle cell, by stretching out, and does not grow to confluency
as normal MDCK II cells. One plausible explanation for this, is integration of the vector into a
gene that has been inactivated.
The variable serglycin-His-flag mRNA expression levels among the clones expressing the
recombinant molecule, is probably due to the site of incorporation into the genome. Factors as
chromatin structure and methylation of the area surrounding the inserted vector, are likely to
influence the accessibility for transcription of the inserted gene. It could also be that some
subclones have integrated multiple copies of the transfected vector into the genome.
There is considerable evidence that the 250 kDa PG analysed represents the recombinant
serglycin-His-flag. The serglycin-His-flag mRNA expressing clones 1-7, 1-10 and 4-2 were
shown to secrete a 250 kDa PG both to the apical and basolateral media (figure 4-12.B), which is
absent in non-transfected clones. This 250 kDa PG also binds with high affinity to the Ni2+
immobilised Hi-Trap chelating column (figures 4-20.C&D). Tram Thu Voung (1998), has also
previously shown that this band is precipitated in immmunoprecipitation experiments, with
antibodies against the His-flag. These results together highly favours that the 250 kDa PG
represents the serglycin-His-flag gene product. An attempt to identify the 250 kDa PG band with
an anti-His-flag antibody did not succeed, due to problems with transfer of the 250 kDa PG onto
a blotting membrane, since the highly negatively charged GAG chains do not bind to most
protein binding membranes. Instead a cationic membrane (Hybond-N+) was used for electricalblotting in SSC. Unfortunately, the background was to high, and specific detection failed (result
not shown). Recently, a new dot blot assay for quantitation of GAGs has been published
(Bjornsson, 1998), where binding of proteoglycans can be increased by wetting the membrane in
cationic detergents prior to blotting (Karlsson and Bjornsson, 1998). This dot blot assay can be
used to optimise a western blot system for detection of the serglycin-His-flag.
130
5 Discussion
The serglycin-His-flag mRNA expression level for the different subclones, did not correlate with
the serglycin-His-flag secretion to apical and basolateral media. The relative secretion of
serglycin-His flag, as percentage of the total secretion of 35(S)proteoglycans, was determined to
be 45 percent (apical) and 43 percent (basolateral), respectively, for clone 1-7. The same values
for clone 4-2 were 32 percent (apical) and 35 percent (basolateral), respectively (table 4-9). This
result does not correlate with the mRNA expression levels for the transfected clones, since clone
4-2 was shown to express more serglycin mRNA than clone 1-7 (figures 4-11.B and 4-12.A).
The reason for this is not investigated in detail. However, it is important to note that the MDCK
II cell line consists of a heterogeneous cell suspension, and that the different isolated subclones
therefore may show minor differences in their phenotypes. The different clones can vary in
proteoglycan core protein synthesis, in basal GAG synthesis, and sulphation efficiency. A
variation in proteoglycan synthesis is indeed observed between the two established MDCK cell
strains. The MDCK I cell line secretes 58 % CS and 44 % HS, whereas the MDCK II cell line
secretes 31 % CS and 69 % HS (Svennevig et al., 1995). Since the MDCK I cell line is derived
by selective culturing of the MDCK II cell line (Simmons, 1981), the MDCK II sub clones may
also vary in proteoglycan expression.
Another factor, which may influence on the secretion of the recombinant serglycin-His-flag, is
the culturing time prior to metabolic labelling. In the beginning the cells were usually labelled for
20 hours after three days growth on Transwell polycarbonate filter membranes (performed by
Tram Thu Voung). However, in all labelling experiments present in this report, the cells were
labelled after four days (all other procedures were identical). Thus, it is interesting that Tram Thu
Voung found clone 1-10 to have the highest 35(S)macromolecule secretion, while clone 1-7
shows the highest secretion level in all experiment in this report. In an experiment where the
proteoglycan synthesis in clone 1-7 was tested by labelling with 35(S)sulphate for 20 hours 2, 3
and 4 (normal) days after seeding onto the filter membranes, the proteoglycan synthesis was
reduced gradually (result not shown). Thus, it might be that the different subclones have variable
reduction in proteoglycan synthesis during development of the polarised cell layer. To
characterise proteoglycan synthesis in the subclones more, the experiment above should be
performed for all subclones, including the wild type MDCK II cell line.
The different subclones did also show minor differences during the culturing, which may be an
effect of the transfected serglycin-His-flag. Subclone 1-10 was usually found to reach confluency
first, when cultured in 75 cm2 flasks, followed by clones 4-2, 1-7 and 7-4. This was not due to a
higher cell number in the culture flask, which actually was found to be lower in a confluent layer
for clone 1-10 than for the other clones (result not shown). It thus seems that a high serglycin
synthesis stimulates cell division and formation of the confluent cell layer. Clone 1-10, also
seems to have a reduced endogenous proteoglycan synthesis, compared to the other tested
subclones (figure 12.B). It may be that a high expression level of serglycin-core protein reduces
the possibility for GAG attachment and sulphation of the wild type proteoglycans. The serglycin
core protein could in this clone function similarly to xylosides, which prime GAG synthesis and
reduce the attachment of GAGs to the endogenous proteoglycan core proteins (Kolset et al.,
1990). In that chase, the serglycin-His-flag core protein is likely to influence both HS- and CSGAG synthesis. Thus, the growth stimulating effect may be caused by reduced GAG attachment/
sulphation of wild type proteoglycans, and/or the high presence of serglycin-His-flag. For the
reasons above, clone 1-10 was found to differ from the wild type MDCK II cell line, and was not
used to study the polarised secretion of the serglycin-His-flag.
131
5 Discussion
The presence of serglycin-His-flag in intracellular fractions was not analysed, since the aim of
the study was to study the secretion of the serglycin-His-flag. However, the high presence of
recombinant serglycin-His-flag extracellularly, indicated that most was secreted. A pulselabelling experiment could confirm this, but was not performed. Even though serglycin is stored
in intracellular granules (with bound proteases) and released by stimuli in mast cells (Hunt et al.,
1997), it is unlikely that the epithelial cell line MDCK II could store the serglycin-His-flag
intracellularly in a similar way. In one experiment, the total 35(S)macromolecules in the apical,
the basolateral and the cell fractions was counted (appendix 1.2). This experiment revealed that
the intracellular PG in serglycin-His-flag mRNA producing clones, increased proportional with
the apical and the basolateral secretion.
5.2.2 Optimising the method for Ni2+ chelating chromatography
When starting to optimise the method for purification of serglycin-His-flag by metal chelating
chromatography, only two studies were found that used his-flag fusion protein for isolation of
proteoglycans. A recombinant decorin fusion protein (Ramamurthy et al., 1996) and a
recombinant biglycan fusion protein (Hocking et al., 1996) were both isolated by nickel chelating
chromatography. However, purification of these two proteoglycans was considered to be simpler,
because these small proteoglycans have only one and two GAG-attachment sites, respectively,
and both are modified with chondroitin/dermatan sulphate glycosaminoglycan chains in vivo. In
addition, the proteoglycans were expressed in HT-1080 cells, derived from fibrosarcoma, which
has a low HS synthesis. Serglycin, on the other hand, contains 8 attachment sites, arranged in
close position in the middle of the core protein (Humphries et al., 1992), and was found to be
modified with both CS and HS when expressed in the MDCK II cells (figure 4.13). The
domination of glycosaminoglycan chains, compared to core protein in the serglycin-His-flag,
could make the isolation of this proteoglycan more difficult. The basal HS synthesis in the
MDCK II cell line is also high, as mentioned before, which further could influence on the
isolation of the recombinant protein.
Previous studies had shown that CSPG does not attach to either Co2+ or Cu2+ immobilised matrix
during isolation of perforin and granzymes from cytotoxic lymphocytes (Winkler et al., 1996),
and that re-injected decorin after removal of the His-flag by protease, did not attach to the Ni2+
column (Ramamurthy et al., 1996). CS-GAG chains were thus not expected to interfere with the
system. Meanwhile, it was not known if the high density of the GAG chains, or the presence of
HS-GAG chains attached to the core protein, would influence on the purification of the
recombinant serglycin-His-flag. Thus, there was a need to optimise the conditions for proper
purification, to be able to purify the recombinant serglycin-His-flag.
In the pilot experiments, where elution of the bound serglycin-His-flag was facilitated with
reduction in pH or increasing imidazole concentrations in the buffer, it was found that some
35
(S)macromolecules secreted from the moc-transfected clone 7-4 bound with low affinity to the
column (figure 4-15). The unspecifically bound 35(S)macromolecules were later characterised as
HS proteoglycans normally expressed in the wild type MDCK II cell line, and were found to
have a large molecular weight, since they did not enter the gel during SDS-PAGE (figure 420.C&D), and eluted in V0 during the Superose 6 gel filtration analysis (figures 4-21 and 4-22).
It is not fully determined if the HS-GAG chains in this proteoglycan mediate the unspecific
binding. This could be tested by -elimination, where the GAG chains are chemically cleaved off
from the core protein, prior to purification with the Hi-Trap chelating system. However, we
132
5 Discussion
found this analysis unnecessary, since other HS proteoglycans did not attach to the column. Thus,
it seems unlikely that HS-GAG chains alone are able to mediate binding to the column, but rather
the proteoglycan binds by another motif, or several motifs working together. This is also
supported by the observation that it was not possible to separate the unspecifically bound
proteoglycan from the specifically bound serglycin-His-flag by the reduction in pH, even if it
could be achieved with increasing imidazole concentration. Most likely, the unspecifically bound
proteoglycan contains two or more histidines in a proximal position, which are able to mediate a
weak interaction with the column compared to the high affinity mediated by the six repeated
histidines in the recombinant serglycin-His-flag. The histidines in both molecules are likely to
accept protons within the same pH range, and thus both molecules will elute with the same pH.
In an attempt to reduce the unspecific binding, the Ni2+ ions immobilised on the Hi-Trap
chelating column were exchanged with Co2+. The bound material was not analysed, but less
material in the high affinity fraction indicated that recombinant serglycin-His-flag either was not
bound to the column, or was eluted in the low affinity fraction (figure 4-17). Thus, Co2+ ions are
not suitable for purification of serglycin-His-flag. Other ions were not tried out, but it may be that
other ions are better than Ni2+ ions, for instance Cu2+ ions, which are frequently used in metal
chelating chromatography. Immobilised Cu2+ ions should mediate stronger binding, according to
the common Cu2+ > Ni2+ pattern (Sloane et al., 1996). However, increased specific binding can
also be followed by increased unspecific binding, and thus, it will not necessarily increase the
purity of the isolated serglycin-His-flag.
The serglycin-His-flag bound with both low and high affinity to the Hi-Trap chelating column.
Except for more CS-GAG attached to the serglycin-His-flag in the high affinity peak, other
structural differences between the low and high affinity fractions were not found. The retention
values are nearly identical for both variants (peak 1: Kav= 0.25, peak 2: Kav= 0.28), which should
be considered as not significantly different, due to the broad peaks (Kav= 0.1 to 0.5 for both, see
figures 4-21 to 4-24). The molecular weight of the eluted serglycin-His-flag, in both the low and
high affinity fractions is thus nearly identical. Nevertheless, for unknown reasons, the serglycinHis-flag modified with HS seems to mediate a weaker binding to the Hi-Trap chelating column
than the variant modified with CS. This may also be considered as another prove that the heparan
sulphate chain in the unspecific bound proteoglycan, not are responsible for the unspecific
binding to the Hi-Trap chelating column, as indicated above.
The observation that the serglycin-His-flag isolated from polarised MDCK II cells seems to have
a lower affinity for the Hi-Trap chelating column is not investigated further (compare figures 415.D and 4-18). However, a reasonable explanation is that the heparan sulphate synthesis is more
active in non-polarised cultures of MDCK II cells.
Some of the experiments done during the optimising of the Hi-Trap chelating column system,
also indicated that the binding of serglycin-His-flag varied with the ion concentration in the
running buffer. The binding of HS-GAG modified serglycin-His-flag may be reversly related to
the ion concentration in the running buffer. In an experiment identical to the one presented in
chapter 4.5.2, with the exception that the running buffers contained 500 mM NaCl instead of 150
mM, more serglycin-His-flag eluted in peak 1 and less serglycin-His-flag eluted in peak 2. In this
experiment, the serglycin-His-flag eluted in the peak 1 fraction was modified with HS-GAG only
(both apical and basolateral), whereas serglycin-His-flag eluted in peak 2 was modified with both
CS- and HS-GAG (result not shown). This indicates that increased ion concentration, favours
separation of CS- and HS-serglycin-His-flag. However, unspecifically bound proteoglycans were
also eluted in peak 1, when 500 mM NaCl was present in the running buffer. Thus, low ionic
133
5 Discussion
strength in the running buffer seems to facilitate more pure serglycin-His-flag eluted in peak 2,
whereas high ionic strength seems to increase the ratio of CS- compared to HS-serglycin-His-flag
in peak 2.
In the initial experiments, low recovery after Hi-Trap chelating chromatography was observed
when aliquots of the samples was counted in a scintillation counter. This was not observed when
the samples were collected directly in scintillation tubes and the whole fraction counted. It was
later found that the proteoglycans adhered to the collecting tube wall when eluted from the HiTrap chelating chromatography (detected by the experiment in appendix 3). Binding of PGs to
the tube wall was not observed for eppendorf tubes, which were used for collecting of sample in
all experiments presented in his thesis.
5.2.3 Glycosaminoglycan attachment on serglycin-His-flag core protein
The MDCK II cells were transfected with the recombinant serglycin-His-flag to study the
secretion of an easily detectable chondroitin sulphate proteoglycan in MDCK II cells. Serglycin
has almost exclusively been reported to be a CS proteoglycan (Kolset and Gallagher, 1990), with
the exception of a CS and heparin hybrid in mouse mastocytoma cells (Lidholt et al., 1995). We
therefore expected the serglycin-His-flag to be modified with chondroitin sulphate chains when
expressed in MDCK II cells. The glycosaminoglycan analysis, however, revealed that the
serglycin-His-flag was modified with both CS and HS. In clone 1-7, the secreted serglycin-Hisflag was modified with ~40 percent CS and ~60 percent HS, whereas the same values for clone
4-2, were ~30 percent CS and ~70 percent HS (table 4-10).
The HS-GAG attachment on the serglycin-His-flag core protein in the MDCK II cells, indicates
that the type of GAG chain attached to a GAG attachment site in a proteoglycan is dependent of
several factors. In which way the cell is able to decide the attachment of a particular type of GAG
to an attachment site, is not fully clarified. Several factors have been postulated to influence what
type of GAG that is elongated on the linkage region. These include (a) the amino acid sequence
flanking the serine residue, (b) the access of UDP-sugars and the presence of GAG synthesising
enzymes in the Golgi apparatus, and (c) phosphorylation or sulphation of the linkage region (see
introduction). The presence of both CS- and HS-GAG chains attached to the same type of core
protein, also makes it possible that the proteoglycan is secreted as a hybrid, consisting of both
CS- and HS-GAG chains on the same core protein. As mentioned above, the serglycin core
protein is secreted as a CS proteoglycan in hematopoietic cells, or as a hybrid consisting of both
CS and heparin in mouse mastocytoma cells, which proves that such hybrids can be synthesised.
Serglycin contains a typical HS assembly site
To try to find out why the serglycin-His-flag core protein was modified with HS-GAG chains in
MDCK II cells, we examined the GAG attachment site in serglycin and compared it with HS
assembly sites in other HSPG. The GAG attachment region of serglycin, which contains a
multiple Ser-Gly region flanked by acidic acids (complete amino acid sequence for serglycinHis-flag in appendix 1), was indeed similar to what is suspected to be the consensus GAG
attachment site for heparan sulphate assembly (Zhang et al., 1995). Thus, since serglycin contains
a preferred HS attachment site, the observation that the serglycin core protein is found to be
modified with CS-GAG in most cells studied, indicates that the attachment of HS-GAG may be a
cell specific phenomenon.
134
5 Discussion
Attachment with another type of GAG than CS on the serglycin core protein is described only
once, when secreted as hybrid, consisting of both CS and heparin, by mouse mastocytoma cells
(Lidholt et al., 1995). However, since heparin and HS only differ in the amount of epimerisation
and sulphation, the polymerisation of the disaccharides is probably initiated by the same
transferases, and both initially synthesised as HS-GAG chains. This, together with our results,
clearly demonstrates that the GAG attachment region alone, can not be the only factor that
determines which type of GAG to be synthesised on a core protein. In some way, also the GAG
synthesising machinery in the Golgi apparatus determines the type of GAG chains to be attached
on the core protein.
The CS- and HS-GAG attachment on the serglycin-His-flag shows that the same attachment
region can be modified with both types of GAG chains in the same cell. However, this is not
observed for all GAG attachment sites. Syndecan-1 contains two GAG attachment regions,
where the N-terminal primes both CS-GAG and HS-GAG attachment, whereas the other primes
CS-GAG attachment only, when expressed in Chinese hamster ovary cells (Zhang et al., 1995).
The observation that HS sites always are capable to prime CS synthesis, but that some sites
primes CS synthesis only, is probably an important control point. The cell can avoid HS
assembly and prime CS assembly on an HS site, simply by repressing the HS synthesis. This may
also explain why serglycin mostly has been described as a CSPG. The proteoglycan has so far,
with one exception, not yet been studied in cells able to prime HS synthesis. If the hematopoietic
cells control the repression of the HS synthesis with absence of heparan sulphate transferases in
the Golgi apparatus, or other factors, for instance inhibition of transport of UDP-GlcNAc into the
Golgi lumen, should be interesting to know.
Serglycin is secreted either as a CS or as a HS proteoglycan
The observation that serglycin-His-flag is modified with both CS- and HS-GAG also render it
possible that the same core protein is secreted as a hybrid, modified with both GAG chains. A
fraction of the serglycin is secreted as a hybrid, consisting of both CS and heparin, in mouse
mastocytoma cells (Lidholt et al., 1995), which indicates that the core protein may prime hybrids.
Hybrid proteoglycans can be detected by degradation of either the CS-GAG or the HS-GAG
chains attached to the protein core, which results in reduced molecular weight of the remaining
molecule. This shift in the molecular weight of the remaining proteoglycan can sometimes be
detected by both gel filtration and PAGE analysis, as observed for a cell surface hybrid
proteoglycan made by mouse mammary epithelial cells (Rapraeger et al., 1985). This hybrid
proteoglycan, with a total molecular weight ranging from 130-260 kDa, was estimated to contain
one or two CS-GAG chains for every fourth HS-GAG chains.
The experimental procedures used in this thesis are not extensive enough to fully conclude if the
serglycin-His-flag synthesised and secreted by MDCK II cells is a hybrid consisting of both CSand HS-GAG chains, or separate CS and HS proteoglycans. So far, however, no results favour
the existence of a hybrid, and thus it seems that the core protein is modified with either CS- or
HS-GAG chains. The serglycin-His-flag eluted in peak 1 consists of 20-30 % CS and 70-80 %
HS (apical) and 100 % HS (basolateral) (table 4-8), which proves that some serglycin-His-flag
core proteins are attached with HS chains only. The serglycin-His-flag eluted in peak 2 contains
55-65 % CS and 45-35 % HS, and thus a hybrid serglycin-His-flag would probably contain
approximately the same amount of both CS- and HS-GAG chains attached to the same core
protein. This would reduce the molecular weight from 250 kDa to about the half after
degradation of one type of GAG chains. Neither the SDS-PAGE (figure 4-13) nor the Superose 6
gel filtration analysis (figures 4-21 to 4-24) of chondroitinase ABC lyase or nitrous acid treated
samples, showed indication of a reduced molecular weight of the remaining proteoglycan after
135
5 Discussion
degradation of one type of GAGs. The serglycin-His-flag eluted with Kav0.25 both before and
after treatment. In comparison, gel filtration analysis of free GAG chains attached to the
serglycin-His-flag (released by -elimination) eluted with Kav=0.5-0.6 (result not shown). These
free GAG chains are estimated to have a molecular weight larger than 11 kDa, by comparison
with the GAG chains attached on the serglycin secreted by U937 cells (Mustorp, 1997).
However, the possibility of a hybrid serglycin-his-flag can not yet be excluded, and should be
examined further, for instance by anion exchange chromatography. Since the CS- and HS-GAG
show different affinity for anion exchangers, the purified serglycin His-flag is expected to elute
in two peaks if it contains separate GAG chains, and in a single peak if it is a hybrid
proteoglycan. Moreover, degradation of the chondroitin sulphate chains in a hybrid, with
chondroitinase ABC lyase treatment, is expected to reduce the remaining proteoglycans affinity
for the anion exchanger1.
As mentioned previously, the serglycin core protein contains 8 attachment sites, arranged in close
position in the middle of the core protein (Humphries et al., 1992). It would be interesting to
know if some serines in the attachment site in serglycin-His-flag show a preferred attachment for
either CS- or HS-GAG in MDCK II cells. Even if all serines are potential attachment sites, it is
not fully documented that all serines are modified with GAG chains. However, no known good
technique exists, which could be used to examine this, since the repeating Ser-Gly attachment
region is shown to be resistant to proteases (Stevens et al., 1985). For the same reason,
determination of preferred CS- and HS-GAG attachment sites in the GAG-attachment region is a
great challenge.
Factors with may determine either CS- or HS-GAG synthesis on the core protein
The fact that serglycin is secreted as a hybrid by mouse mastocytoma cells (Lidholt et al., 1995),
proves that such hybrids exist. Since hybrids seems to be absent in MDCK II cells, there must be
some differences in the glycosaminoglycan synthesis machinery between MDCK II cells and
mouse mastocytoma cells.
It could be that the synthesis of one type of GAG chain excludes the possibility of modification
with the other type of GAG chain in MDCK II cells. The cell could control this by having
separate membrane locations for the CS and HS synthesis machinery in the Golgi apparatus, as
hypotheses by Silbert and Sugumaran (1995). Transport of the proteoglycan core protein to a site,
which contains GlcNAc transferase I, or alternatively, GalNAc transferase I, would be a critical
process. Certain sequences in the core protein, or modifications such as sulphation and
phosphorylation of the linkage region, could regulate this transport. The linkage region in
proteoglycans from various sources has been studied, which has shown that C-2 of xylose is a
major site for phosphorylation in both chondroitin sulphate (Oegema et al., 1984; Sugahara et al.,
1992) and heparan sulphate proteoglycans (Fransson et al., 1985). Structural studies have also
revealed that sulphation of C-6 of both Gal residues and C-4 of Gal adjacent to GlcA was
characteristic for chondroitin sulphate (de Waard P. et al., 1992; Sugahara et al., 1988; Sugahara
et al., 1991; Sugahara et al., 1992), whereas, sulphation of the two Gal residues in the linkage
region was not found in heparin or HS proteoglycans (Sugahara et al., 1992; Sugahara et al.,
1994). Sulphate and phosphate has never been found in the same linkage region. Thus,
1
For unknown reasons, the chondroitin sulphate chain mediates a stronger binding to an anion exchanger than the
heparan sulphate chain, even though the chondroitin sulphate chain is generally less negatively charged.
136
5 Discussion
sulphation of Gal or phosphorylation of xylose in the linkage region can be signals for specific
GAG synthesis, by targeting the core protein to the right membrane location.
The secretion of the recombinant serglycin-His-flag in MDCK II cells would be an excellent tool
for studying this, by investigating potential differences in the linkage region between the CS- and
the HS-GAG modified serglycin-His-flag. It would be interesting to know if any of the galactoses
in the linkage region are sulphated, if the xylose is phosphorylated, and especially if there are any
differences in the linkage region of the CS- and the HS-GAG modified serglycin-His-Flag. For
instance, phosphorylation may be required for HS-GAG elongation, which could be prevented by
sulphation of the Gal residues (Figure 5-1). Xylose is generally non-phosphorylated in
proteoglycans derived form tissues with an abundant extracellular matrix (HS proteoglycans)
(Cheng et al., 1996; Sugahara et al., 1995a). However, only the secreted proteoglycans are
analysed, and it is not unlikely that the phosphate is removed prior to secretion, since addition of
the first GlcA in the biosynthesis of decorin is followed by rapid dephosphorylation in fibroblasts
cells (Moses et al., 1997). Phosphorylation could then be necessary for guiding of the
proteoglycans to a site for GAG elongation.
GlcA  Gal  Gal  Xyl  O  Ser
S
P
CS-synthesis CS-synthesis
HS-synthesis
Figure 5-1: Possible role of sulphation and phosphorylation of linkage region.
The transfer of the core protein to separate membrane locations for CS and HS synthesis
machinery in the Golgi apparatus, is not necessarily determined by signals in the linkage region.
The linkage region in both types of GAGs could also be identical, and selection for CS or HS
synthesis mediated by other factors. If so, the selection for GAG synthesis must lie in the HS
synthesis machinery because it is shown that the HS synthesis requires specific structures on the
xylosides, while the CS synthesis is primed on almost all xylosides (Fritz et al., 1994; Fritz et al.,
1997). In addition, one of the GAG attachment sites in syndecan-1 seems to never be modified by
HS-GAG. This site is modified with CS-GAG, both in wild type CHO cells, and in a mutant
CHO cell line (CHO 1d1D) (Zhang et al., 1995). This mutant CHO cell line has a deficiency in
the 4'-epimerase that catalyses GlcNAc  GalNAc formation, and thus makes less CS when
deprived of endogenous GalNAc (Esko et al., 1988). Still, with minimal CS synthesis, one of the
GAG attachment regions in syndecan-1 never primes HS synthesis, which indicates that HS
synthesis seems to be more controlled than CS synthesis.
137
5 Discussion
Without signals in the linkage region, one possible way to prevent CS-GAG assembly on HSGAG sites, is to locate the site for HS synthesis more proximal in the Golgi apparatus than that
for CS synthesis. As the core protein is transported through the Golgi apparatus, the linkage
region is modified with HS-GAG if it contain an appropriate HS-GAG assembly site, and if not,
the core protein is passed on to the site for CS synthesis. Indeed, treatment with BFA has
revealed that the HS assembly is primed more proximal in the Golgi apparatus than CS synthesis
in both rat ovarian granulosa cells (Uhlin-Hansen and Yanagishita, 1993) and in melanoma cells
(Spiro et al., 1991). Separate Golgi localisation of the CS and HS machinery has not been
determined in MDCK cells, because MDCK cells and PtK cells (also a kidney epithelial cell line)
are resistant to the drug BFA (Ktistakis et al., 1991; Sandvig et al., 1991). However, it is thought
that the epithelial cell line MDCK II also has separate Golgi localisation of the CS and HS
synthesis machinery, since sulphation of CS and HS has different sensitivity to the inhibitor
chlorate, which inhibits the formation of PAPS (Prydz, personal communication).
Thus, the different membrane localisation of the CS and HS synthesis machinery, may directly
function to avoid hybrid modification of typical HS attachment regions. In this context, it is very
interesting that the mouse mastocytoma cell line, found to secrete hybrid serglycin with both CSGAG and heparin chains (Lidholt et al., 1995), is found to have co-localised CS and HS
synthesis machinery (Uhlin-Hansen et al., 1997). Even with different membrane localisation of
the CS and HS synthesis enzymes, a hybrid could be formed, however, if the core protein has a
poor HS-GAG attachment region, which then will be non- modified at the HS assembly site, and
first modified when the core protein reaches the site for CS assembly. A proximal localisation of
the HS synthesis enzymes in the Golgi apparatus, may thus be an important control factor
determining the type of GAG to be synthesised on the core protein.
This model does not, however, explain why the serglycin-His-flag core protein is modified with
both CS-GAG and HS-GAG in MDCK II cells. Since serglycin contains a HS-GAG assembly
site, it would be expected that all the serglycin-His-flag core proteins were primed with HSGAG. However, it could be that, in addition to have a strict location of the enzymes involved in
the GAG synthesis in the Golgi apparatus, the enzyme concentrations, or the amount of the UDPsaccharides in the Golgi lumen, also are strictly controlled. The MDCK II cell could control the
efficiency of the GAG synthesis machinery by regulating the transport of the different UDPsaccharides into the Golgi lumen by the activity of the UDP-translocators (Hirschberg et al.,
1998). Thus, with a high core protein content in the Golgi apparatus, the availability of either
UDP-GlcNAc or the different HS synthesising enzymes may be less than required to assemble
HS-GAG on all HS sites.
Thus, it is very interesting that, even if the analysis of the GAG-attachment in clone 1-7 and 4-2
revealed that clone 1-7 secretes more serglycin-His-flag than clone 4-2, the HS-GAG
modification in both clones was nearly identical (table 4-9). It could be that the CS synthesis
machinery is more dominant clone 1-7, but it is also possible that the HS synthesis machinery is
nearly saturated in both clones, and that the excess core protein is transported to the site of CSGAG attachment. A saturation of the HS synthesis machinery in clone 1-7 and 4-2 is not
unlikely, since the wild type PG synthesis was reduced in the high serglycin-His-flag expressing
clone 1-10, even if this clone secreted less 35(S)macromolecules than both clone 1-7 and 4-2
(figure 4-12.B). The CS- versus HS-GAG attachment in clone 1-10 is not analysed, but it should
be interesting to know if this clone primes even more CS-GAG on the serglycin-His-flag than
clone 1-7. Another factor facilitating a possible saturation of the HS synthesis machinery, is that
the MDCK I cell line, which is found to secrete more proteoglycans into the medium than
138
5 Discussion
MDCK II, also primes more CS proteoglycans (Svennevig et al., 1995). A tight control of the HS
synthesis machinery may thus be important for the CS- versus HS-GAG assembly in MDCK
cells. If this is controlled by the different chain elongating enzymes, or the concentration of
GlcNAc in the Golgi apparatus should be interesting to know.
With a nearly saturated HS synthesis machinery, it could be anticipated that this would increase
the possibility of hybrid formation, and thus hybrid serglycin-His-flag should be present.
However, this is not necessarily true. The serglycin core protein has a highly concentrated
glycosaminoglycan attachment site, with the eight sites within an 18 amino acid peptide sequence
(Humphries et al., 1992). Recognition and capture of the core protein to the location of the HS
synthesis machinery is thus likely to be followed by GlcNAc transfer to all linkage regions
synthesised on the core protein, followed by further elongation of HS-GAG. So, if all HS
assembly sites in the Golgi apparatus are occupied, the core protein will escape and reach the site
for CS synthesis, and thus no hybrids are formed. The different factors, which may determine the
type of GAG attached to the core protein is summarised beneath (Figure 5-2).
TGN
CS-synthesis
4
3
2
HS-Synthesis
1
Formation of
linkage region
Figure 5-2: Model for factors involved GAG attachment in MDCK II cells.
The linkage region is formed, as the core protein is transported through the Golgi apparatus. (1): During this
process, the linkage region may be modified with signals that facilitate either CS or HS polymerisation on the
linkage region (phosphorylation of Xyl, sulphation of Gal). (2): When the proteoglycan reaches the site for HS
synthesis, the linkage region may be polymerised with HS-GAG. HS-GAG assembling requires that the core
protein contains a HS assembling site, phosphorylation of Xyl may be required, and UDP-GlcNAc, UDP-GlcA
and HS polymerising enzymes must be available. (3): Then the core protein reach the site for CS synthesis,
where all linkage regions not modified with HS-GAG, are polymerised with CS-GAG. Sulphation of the linkage
region may prevent HS assembly or facilitate CS synthesis. (4): After the polymerisation is initiated, the core
protein is sulphated (and epimerised for HS) in various positions. These modifications occur simultaneously
with the polymerisation reactions.
139
5 Discussion
140
5 Discussion
5.2.4 Polarised secretion of the recombinant serglycin-His-flag in MDCK II cells
It is important to understand the molecular mechanisms that govern sorting of cell proteins, since
the distribution of the proteins is important for the function of the cells. The importance of
receptor localisation and functions in signal transduction, is observed in several diseases, which
are caused by changes in receptors localisation or mutations in sorting determinants. A lack of
sorting signal may have fatale consequences. In familiar hypercholesterolemia, the low density
lipoprotein receptor is unable to internalise, and can not transport cholesterol for storage as
cholesterol esters (Brown and Goldstein, 1986). Lysosomal storage disease is caused by genetic
defects that effect one or more of the lysosomal hydrolases, and results in accumulation of their
undigested substrates in the lysosomes. In inclusion cell (I-cell) disease, the missorting of the
hydrolytic enzymes has been traced to a defective or missing GlcNAc-transferase, which
phosphorylates the hydrolytic enzymes in the cis-Golgi. Thus, the non-phosphorylated lysosomal
enzymes are not recognised by the M6P receptor, and not packaged into lysosomal transport
vesicles in the trans-Golgi network (Alberts et al., 1994).
The study of mechanisms underlying the molecular sorting of some molecules, has been
intensified during the last decade, due to the discovery of the first sorting signals. The now well
established apical targeting signal, the GPI-anchor, was first demonstrated to have this function
by Brown et al. (1989). GPI-anchored proteins are mainly localised to the apical plasma
membrane in MDCK II cells (Lisanti et al., 1988), and are also included in the detergent-resistant
sheets ("rafts") formed in the TGN (Brown and Rose, 1992). Recently, it has however become
clear that the GPI-anchor may not be an apical targeting signal, as previously expected.
Examination of GPI-anchored proteins found to be apically secreted in previous experiments, has
revealed that all of them contained N-liked saccharides, the another characterised apical targeting
signal (Gut et al., 1998). Removal of these saccharides from the core proteins, result in nonpolarised secretion of the GPI-anchored proteins (Benting et al., Simons lab, unpublished data).
The role of GPI-anchor in apical targeting is thus unclear, but it may be important for anchoring
of the protein near the membrane, which then could facilitate the packaging into the apical
transport vesicle.
Recent models for apical transport, should thus be restricted to employ N-glycosylation as the
sorting determinant, and sphingolipid-cholesterol "rafts" as platforms (Simons and Ikonen,
1997). "Rafts"-association is found for several apically targeted molecules, like influenza virus
HA, in MDCK II cells (Scheiffele et al., 1997). A lectin-like protein, VIP-36, was isolated from
apical transport vesicles and found to associate with "rafts" (Fiedler et al., 1994).
Characterisation of VIP-36 revealed that the molecule was able to bind Ca2+ and GalNAc
(Fiedler and Simons, 1996), and was thus, initially by us, suspected as a candidate for binding
and targeting of CS to the apical surface in MDCK II cells. However, further experiments have
revealed that the binding-coefficient for complex-formation between VIP-36 and GalNAc is too
high for having any biological relevance (Fullekrug et al., unpublished data).
In MDCK II cells, chondroitin sulphate chains are secreted to the apical medium, but not to the
basolateral medium. The observed apical targeting of CS and a dominating basolateral targeting
of HS in MDCK II cells, indicates that the proteoglycans have functions on opposite sides of the
epithelial layer. This may be due to the physiological function of the kidney epithelium. As
mentioned in the introduction, the epithelium of the distal and proximal tubules is involved in the
filtration and re-absorption of nutrients in the kidney (figure 1.2). One of the major properties of
the basal lamina is to prevent leakage of charged molecules between the blood and the filtrate.
Reduced sulphation of HS proteoglycans, or altered GAG attachment in the basal lamina of the
141
5 Discussion
kidney, often leads to dysfunctional basal lamina (Kasinath, 1995; Kasinath et al., 1996; Kolset
et al., 1994). Increased delivery of CS proteoglycans, which are less sulphated, and thus, less
negatively charged than HS proteoglycans, may also reduce the charge specific repulsion of the
basal lamina. Thus, exclusion of CS proteoglycans from basolateral secretion may be important
for the formation and the maintenance of the basal lamina.
The wild type proteoglycans secreted by the MDCK II cell line have not been well studied, but
most HS proteoglycans secreted basolaterally are presumably matrix components. Antibodies
against perlecan and versican revealed that both proteoglycans were secreted by MDCK II cells
(Svennevig et al., 1995). Perlecan (Mw ~470 kDa) is a basement membrane proteoglycan (HS),
and is in part responsible for the charge specific ultrafiltration of this extracellular matrix.
Versican (CS) (Mw ~260 kDa) contains domains that are highly homologous to aggrecan, and
may play a role in intracellular signalling, cell recognition and to connect extracellular matrix
components and cell surface glycoproteins (Ayad et al., 1994). Most versican was found to be
cell associated, while perlecan was mostly secreted. However, apical transport of both of these
proteoglycans was lower than the basolateral (Svennevig et al., 1995).
In the first experiments, to characterise the proteoglycans secreted by the two MDCK strains, if
was found that the MDCK II cell line secreted a low amount of CSPG to the basolateral
compartment (Svennevig et al., 1995). Later experiments, with 35(S)sulphate labelling of
polarised cultured MDCK II cells, have, however, revealed that the secreted CS proteoglycan(s)
is sorted to the apical compartment exclusively (Vuong, 1998). Variations in growth conditions
were found to not influence on the apical sorting of CS proteoglycan(s), which indicated that the
apical sorting was a stable phenomenon. Why CS was found basolateral in the first experiments,
is still not known.
Treatment of MDCK II cells with hexyl-thio--D-xylosides, which primes CS synthesis
efficiently in MDCK II cells, also results in apical secretion of the major free CS-GAG
synthesised (Kolset et al., 1999). Thus the CS-GAG chain may contain a signal for apical
targeting in MDCK II cells, which is not unlikely since also other saccharides may facilitate in
apical targeting. It is now well documented that N-glycans presumably function as an apical
sorting signal (Scheiffele et al., 1995), and in addition that O-glycosylation also might facilitate
apical targeting in MDCK II cells (Zimmer et al., 1997). It is still not known if the apical
secretion of CSPG in MDCK II cells is independent of "rafts" and glycolipids. Some experiments
indicate this, since treatment with mannosamin and fumonisin B1, which inhibit GPI-anchor
formation and glycolipid synthesis, respectively, did not affect the apical secretion of the CS
proteoglycan(s) (Vuong, 1998). Apical secretion without "rafts"-association has also been found
for other molecules. Both the N-linked and O-glycosylated bovine enteropeptidase (Zheng et al.,
1999), and the non-glycosylated CD3- are delivered to the apical surface without integration
into "rafts" in MDCK cells (Alonso et al., 1997). The previous results, thus indicate that the
sorting of CSPG to the apical surface, probably is mediated by an alternative non-characterised
sorting machinery. It should however be considered that detection of "rafts"-association is
difficult, and thus the involvement of "rafts" in apical targeting of CS should still not be
excluded.
To study the role of the CS-GAG chain in apical targeting, we transfected the MDCK II cell line
with serglycin-His-flag to determine the secretion of an easily detectable CS proteoglycan. As
mentioned already, the serglycin-His-flag core protein was modified with both CS- and HS-GAG
chains, which complicated the study. Purification of the two variants of serglycin-His-flag
followed by glycosaminoglycan analysis was therefore necessary to determine the secretion of
142
5 Discussion
the CS- and the HS-GAG modified variants of the serglycin-His-flag. This revealed that the
serglycin-His-flag secreted by clones 1-7 and 4-2 were modified with 30-40 percent CS and 6070 percent HS. Approximately 20 % (10 % CS and 10 % HS) was secreted apically, while
approximately 80 % (20-25 % CS and 55-60 % HS) was secreted basolaterally (table 4-10). Even
if clone 1-7 secreted more CS-serglycin-His-flag than clone 4-2, the sorting of the synthesised
CS-serglycin-His-flag was 1/3 apical and 2/3 basolateral for both clones, and the secretion of HSserglycin-His flag was 1/6 apical and 5/6 basolateral.
The glycosaminoglycan analysis presented in table 4-10 is determined from one experiment only.
An exact determination of the CS- versus HS-GAG attachment, and apical versus basolateral
secretion of the CS- and HS-GAG modified form of serglycin-His-flag has not been carried out,
since this would require several repetitions of the experiments for statistical analysis. However,
we found this unnecessary, since both clones 1-7 and 4-2 showed similar secretion distribution,
and a clear indication of basolateral targeting of CS-serglycin-His-flag. The experiment was
repeated once for clone 4-2, which gave a secretion pattern nearly identical to the results
presented in tables 4-9 and 4-10. Basolateral targeting of CS serglycin-His-flag was also
consistently observed, during the optimisation of the Hi-Trap chelating chromatography system.
Thus, basolateral secretion of the CS-GAG modified serglycin-His-flag is a reproducible
phenomenon.
A non-sorted secretion of macromolecules by the MDCK II cells, results in approximately 1/3
apical and 2/3 basolateral transport, depending to some extent on the growth conditions, as
observed for growth hormone (GH) (Scheiffele et al., 1995). Thus, CS-serglycin-His-flag seems
to be unsorted, while the HS-serglycin-His-flag may have a regulated sorting to the basolateral
compartment. An enrichment of HS proteoglycans to the basolateral compartment was also
expected. The distribution of endogenous secreted 35(S)macromolecules from MDCK II cells is
25 percent (apical) and 75 percent (basolateral) (Svennevig et al., 1995). This distribution was
also found for the moc-transfected clone 7-4 (figure 4-12.A). Approximately 70 % of the
35
(S)macromolecules found apically and basolaterally represents CS and HS proteoglycans
(apical: 30 % CS and 70 % HS, basolateral: 100 % HS) (Vuong, 1998). With the values above, it
can thus be estimated that approximately 1/5 of the secreted HS is located apically, and that 4/5
is located basolaterally (Table 5-1). The transport of HS modified serglycin-His-flag is thus
similar to the secretion of wild type HS proteoglycans in the MDCK II cell line. This indicates
that the HS modified serglycin-His-flag follows the normal secretion pattern for HS
proteoglycans synthesised by the MDCK II cell line.
GAG type
Compartment
Wild type secreted GAG *
Secreted serglycin-His-flag
Chondroitin sulphate
Apical
Basolateral
1
~1/3
0
~2/3
Heparan sulphate
Apical
Basolateral
~1/5
~1/6
~4/5
~5/6
Table 5-1: The distribution of endogenous and serglycin-His-flag proteoglycans, modified with
CS- and HS-GAG, secreted by the MDCK II cell line.
(*) Values for wild type proteoglycans are determined by the results in (Svennevig et al., 1995) and
(Vuong, 1998).
143
5 Discussion
It is still not known why the CS modified serglycin-His-flag is non-sorted in MDCK II cells.
However, very little is known about the secreted CS proteoglycan(s) in MDCK II cells, apart
from that their molecular weight are above 250 kDa. Thus, structural differences between the
wild type CSPG and the serglycin-His-flag can be responsible for the non-sorted secretion of the
serglycin-His-flag. The most plausible explanation, is that the tight assembly of CS-GAG chains
on the serglycin-His-flag core protein in some way either disturbs packaging, or is not recognised
by the apical packaging machinery. For instance, apically enriched CS-GAG chains secreted by
xyloside-treated MDCK II cells (Kolset et al., 1999), are much smaller (~14 kDa) than the nonsorted CS-serglycin-His-flag (above 200 kDa), and thus probably also easier to package into
vesicles. Alternatively, the chains can be unavailable for binding to a sorting factor that functions
to guide CS-GAG and CSPG to the apical surface. This factor may contain a GalNAc binding
domain, as the "raft"-associated lectin VIP-36 (Fiedler and Simons, 1996). It is unlikely that VIP36 itself is involved in the apical targeting of CS, as explained previously.
Disturbance of the apical secretion signal is not unlikely, because it is likely to be a weak signal.
It is shown that the amyloid precursor-like protein-2 (APLP2) is transported basolaterally in
transfected MDCK II cells, whether the core protein contains a CS-GAG chain or not (Lo et al.,
1995). This basolateral targeting was caused by a cytoplasmic sorting signal in the core protein,
which demonstrates that the possible apical signal in the CS-GAG chain must have been entirely
restrained. The observation that basolateral sorting signals are stronger than the apical, is general.
In three different membrane proteins, with basolateral sorting signals, also containing N-linked
glycans, the presence or absence of the N-linked sugars did not affect the basolateral delivery.
However, in the presence of N-linked glycans and an absence of basolateral signals, all proteins
were targeted apically (Gut et al., 1998).
Another factor, which can explain the non-sorted secretion of the CS-serglycin-His-flag, is the
possibility of structural differences between the GAG chains attached to the wild type CS
proteoglycans and the GAG chains attached to the CS-serglycin-His-flag core protein. The
chondroitinase ABC lyase enzyme used in the analysis, degrades all modified forms of CS-GAGs
(both CS and DS). Thus, a specific modification, as for instance, a distinct sulphation pattern or a
GlcA to IdoA epimerisation (DS) can be required for binding to the apical transport-factor(s). To
exclude this possibility, the exact glycosaminoglycan composition in both the wild type CSPG(s)
and the serglycin-His-flag must be characterised.
The wild type CS proteoglycan could also be dependent on other signals for apical targeting. For
instance, the core protein may contain N-linked and O-linked glycans, which are thought to be
involved in apical targeting (Scheiffele et al., 1995; Zimmer et al., 1997). In the lack of other
basolateral signals, the proteoglycan is thus likely to be apically secreted. Svennevig et al. (1995)
detected the chondroitin sulphate versican in MDCK II cells. This proteoglycan contains many
potential N-linked sites, and thus versican is likely to contain N-linked sugars in MDCK II cells.
If this proteoglycan is the major CS-proteoglycan secreted by the MDCK II cells, it could be
possible that N-glycosylation contributes to the apical targeting, and that the CS-GAG chain may
not contain an apical targeting signal at all. The N-glycans can, however, not be the only signal
that sorts chondroitin sulphate chains to the apical medium in MDCK II cells, since inhibition of
N-glycans synthesis with tunicamycin, had no effect on the apical secretion of CSPG (Prydz,
personal publication). This, together with the result that CS-GAGs assembled onto xylosides are
apically secreted in MDCK II cells, the chondroitin sulphate chain is still hypothesised to be an
apical sorting signal in MDCK II cells. It should, however, be interesting to know why
endogenous proteoglycans and CS-GAGs are apically secreted, while CS serglycin is not.
144
5 Discussion
Besides the predominant secretion of endogenous CSPG to the apical medium, it is also
interesting that the MDCK II cells transport HSPG mainly to the basolateral medium. The HSGAG chain has been assigned a possible role in basolateral transport by other experiments. It is
shown that HS-GAG chain attachment of the glypican core protein, antagonised the apical
delivery by the N-linked glycans2 (Mertens et al., 1996). Whether this effect is caused by a
dominant basolateral signal in the HS-GAG chain, or by an interruption of incorporation into
apical transport vesicles, is still unknown.
If the serglycin-His-flag is synthesised as a hybrid, the presence of HS-GAGs on the core protein
is thus likely to interfere with the apical targeting signal in the CS-GAG. In that case,
introduction of HS-GAG on the core protein can redirect the molecule from apical to basolateral
destination. It is therefore important to further investigate if the CS-GAG attachment on the
serglycin-His-flag secreted to the basolateral compartment is found on a hybrid proteoglycan,
since a hybrid serglycin-His-flag could explain why CS is found attached to the serglycin-Hisflag secreted to the basolateral compartment. However, due to the lack of indications of a hybrid
serglycin-His-flag, this is probably not the explanation for the randomised secretion of the CSserglycin-His-flag.
5.2.5 Stimulation of MDCK II cells with TGF-
Transforming growth factor- (TGF-) is a potent growth factor, which is shown to increase the
level of CS-GAG on syndecan in mouse mammary epithelial cells (Rapraeger, 1989). High
glucose induced TGF-1 production, is shown to cause a dose-dependent increase in the
production of matrix proteins in porcine glomerular mesangial cells (Kolm-Litty et al., 1998). In
MDCK II cells, TGF- seems to inhibit the formation of tubular structures, and to inhibit
epithelial cell division (Santos and Nigam, 1993), thereby suggesting that TGF- modulates
tubulogenesis, as well as branching, in the kidney. The growth factor, TGF-, may therefore play
an important role in the formation of the nephrones and the basal lamina in the kidney.
To try to find out more about the GAG attachment on the serglycin-His-flag core protein, clones
1-7 and 4-2 were stimulated with TGF- during labelling with 35(S)sulphate. Stimulation with
TGF- increased the secretion of 35(S)proteoglycans to the apical medium, whereas the secretion
to the basolateral medium was unchanged (figure 4-27.A). Glycosaminoglycan determination of
the GAG chains attached to the serglycin-His-flag secreted by clone 1-7, showed that the amount
of chondroitin sulphate type chain was increased from ~40 percent (for non-stimulated cells) to
~55 percent (for stimulated cells) (table 4-12). Moreover, after stimulation with TGF-, the
distribution of the HS-GAG modified serglycin-His-flag was altered from 1/6 apical and 5/6
basolateral to 1/3 apical and 2/3 basolateral.
The experiment was not repeated, so the observed alternation of the glycosaminoglycan
attachment by TGF- stimulation is not validated. However, the experiment indicates that TGF-
is able to alter the proteoglycan synthesis in MDCK II cells, by decreasing the HS-GAG
attachment and increasing the CS-GAG attachment on the non-endogenous core protein.
Increased GAG chain length on the serglycin-His-flag was not observed, as was observed for the
CS- and HS-GAG chains attached to syndecan produced by mouse mammary epithelial cells,
In the summary, the authors conclude that: "…. glycanation of the core protein antagonizes the activity of the apical
sorting signal conveyed by the GPI anchor of this proteoglycan." However, since the studied proteoglycan, glypican,
also contains N-glycans, it is more likely that N-glycan chains mediate the apical signal in this proteoglycan.
2
145
5 Discussion
after stimulation with TGF- (Rapraeger, 1989). The alteration of the GAG attachment to
serglycin-His-flag, is thus probably caused either by a decreased efficiency of the HS synthesis
machinery in the Golgi apparatus, or alternatively, by an increased HS attachment on endogenous
core proteins. The latter may be the case, if the attachment of CS-GAG chains on the serglycinHis-flag core protein is due to a nearly saturated HS synthesis machinery in MDCK II cells, as
described previously. With increased endogenous core protein concentration in the Golgi
apparatus, even more serglycin-His-flag will be able to escape from the HS-attachment site, and
reach the site for CS synthesis.
The glycosaminoglycan chains attached to endogenous proteoglycans were not analysed, which
is necessary to understand more about the effect of TGF- stimulation. However, the increased
apical delivery of 35(S)proteoglycans, must be due to an altered delivery of endogenous
proteoglycans in the MDCK II cell. Even if more serglycin-His-flag is secreted to the apical
medium after stimulation with TGF-, the total secretion of serglycin-His-flag apical and
basolateral is about the same (~42 percent) in both non-stimulated and in TGF- stimulated cells
(compare tables 1-10 and 1-12). Thus, the increased apical delivery is caused by increased
secretion of both serglycin-His-flag and endogenously synthesised proteoglycans.
Since the HS-serglycin-His-flag was more randomly secreted after stimulation with TGF-, the
increased apical delivery may be an effect of decreased basolateral targeting of also endogenous
HS-proteoglycans. This should indeed be investigated further, since a decreased basolateral
delivery of HS-proteoglycans, will probably reduce the contents of HS-proteoglycans in the basal
lamina, which may disturb the formation and the maintenance of the basal lamina. The diabetic
nephropathy observed in some diabetic patients, may thus be mediated by high blood glucose
levels, which stimulates TGF- production (Kolm-Litty et al., 1998), which by an unknown
mechanism, stimulates the epithelial cells in the nephrones to reduce the secretion of HSproteoglycan to the basal lamina. Glycosaminoglycan chain attachment to proteoglycans secreted
by MDCK II cells, cultured in low and high glucose medium, is now under investigation.
146