Publication: Glycomic profiling of developmental changes in bovine testis by lectin histochemistry
and further analysis of the most prominent alteration
on the level of the glycoproteome by lectin blotting
and lectin affinity chromatography
Authors
Manning, J.C. ; Seyrek, K. ; Kaltner, H. ; André, S. ; Sinowatz, F. ; Gabius, H.J.
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Publisher
Murcia : F. Hernández
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DOI
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info:eu-repo/semantics/article
Description
Abstract
The emerging concept of the sugar code
attributes functional significance to oligosaccharides of
cellular glycoconjugates by protein (lectin)-carbohydrate
interactions. Hence it follows that monitoring of glycan
expression (glycomic profiling) is not only valuable to
delineate characteristic (phenomenological) changes in
the cell’s glycosylation but will also come up with the
localization of epitopes with potential in biorecognition.
It is for this purpose that we have set up a panel of 16
markers (plant lectins and a carbohydrate-specific
antibody). The selection met two criteria: a) to be able to
detect the common constituents of natural glycans; and
b) to place emphasis on detection of neutral
carbohydrate units at the spatially accessible branch ends
of glycan chains, which are known to be active as
ligands for endogenous lectins in situ. Next, we
incorporated recent insights into the importance of
epitope clustering to turn less abundant oligosaccharides
into potent ligands into our study design. To be able to
focus on such high-affinity sites, we performed
systematic titration studies aimed at defining the probe
concentration at which carbohydrate-independent
background staining is minimal while still yielding a
clear signal. These requirements were met by marker
concentrations of 1.25-2.5 µ g/ml. Under these
conditions, we defined cell-type- and differentiationdependent
changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive
with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for
Gal/GalNAc presentation and in this group most
prominently with the galactoside-specific lectin from
Viscum album L. (mistletoe). Of interest in this context,
this lectin is known as a potent mitogen and signal
inductor as well as haemagglutinin. The Gal/GalNAcdependent
signals decreased markedly in the course of
development and staining was completely lost in the
case of mistletoe lectin 12 weeks after gestation.
Spermatids of adult testis presented respective glycan
epitopes. In contrast to this developmental course of
staining, endothelial cells either maintained a constant
signal intensity or revealed a signal increase during
development for Gal/GalNAc-specific lectins. Their
binding of concanavalin A and the two phytohaemagglutinins
(PHA-E/L), which were not or only
weakly reactive for gonocytes, served as inherent
activity control. Based on lectin blot analysis with the
mistletoe lectin as the marker which detected the most
prominent change, the glycoprotein patterns from fetal
and adult tissue specimens were qualitatively different,
rendering changes in expression of the protein part of glycoproteins more likely than remodeling a
glycoprotein’s glycan chains. Methodologically, results
of this procedure were compared to data obtained with
lectin affinity chromatography and the combination of
the two procedures. Differences in the profiles were
discovered that can be assigned to the disparate ways to
process the detergent extracts. When access to sample
quantity is limited, as is possible in the case of fetal
tissue, direct lectin blotting is recommended.
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