Novel ‘phage display antibodies identify distinct heparan sulfate domains in developing mammalian lung

Abstract

Heparan sulfate proteoglycans (HSPGs) are essential to respiratory morphogenesis in species as diverse as Drosophila and mice; they play a role in the regulation of numerous HS-binding growth factors, e.g. fibroblast growth factors. Moreover, an HS analogue, heparin, modulates lung growth in vitro. However, it has been difficult to assess the roles of specific HS structures in lung development due to technical barriers to their spatial localisation. Lungs from Sprague-Dawley rats were harvested between E15.5 and E19.5 and immediately fixed in 4 % (w/v) paraformaldehyde (in 0.1 M phosphate-buffered saline (PBS), pH 7.4). Lungs were washed in PBS, cryoprotected with 20% (w/v) sucrose (in PBS), gelatin embedded [7.5% (w/v) gelatin, 15% (w/v) sucrose in PBS], before being covered in Cryo-M-Bed (Bright, Huntingdon, UK) and snap frozen at -40 degrees C. Cryosections were cut at 8 microm and stained with the HSPG core protein specific antibody 3G10 and a HS 'phage display antibody, EW4G2V. 3G10 and EW4G2V immunohistochemistry highlighted the presence of specific HS structures in lungs at all gestational ages examined. 3G10 strongly labelled airway basement membranes and the surrounding mesenchyme and showed weak staining of airway epithelial cells. EW4G2V, however, was far more selective, labelling the airway basement membranes only. Mesenchymal and epithelial cells did not appear to possess the HS epitope recognised by EW4G2V at these gestational ages. Novel 'phage display antibodies allow the spatial distribution of tissue HS to be analysed, and demonstrate in situ that distinct cellular compartments of a tissue possess different HS structures, possibly on the same proteoglycan core protein. These probes offer a new opportunity to determine the role of HS in the pathogenesis of congenital defects such as congenital diaphragmatic hernia (CDH), where lung development is aberrant, and the resulting pulmonary hypoplasia and hypertension are a primary cause of mortality.

Stem Cells, Tissue Engineering and Regenerative Medicine

“…3 Instead, use of the phage display antibodies showed how glycans with various epitopes were distributed unevenly between normal lung and its hypoplastic counterpart. [31][32][33] This provides circumstantial evidence that the glycan profile may be critical for this lesion of development, and is supported in further studies. [36][37][38] Together this emphasizes the need for stem and progenitor biology to pay attention to non-template driven processes in order to obtain a fuller and hence more useful view of their regulation.…”

“…For example, interrogation of hypoplastic lungs for expression of glycan epitopes using phage display antibodies shows significant divergence between normal and hypoplastic lung in not only the airway compartment, but also the pulmonary vasculature. [31][32][33] Lethal pulmonary hypertension is associated with lung hypoplasia. 34,35 These studies support the concept that the glycome is essential for normal airway development.…”

Heparan Sulfate Phage Display Antibodies Identify Distinct Epitopes with Complex Binding Characteristics

“…To circumvent the difficulties of analyzing tissue HS in situ, HS-specific single chain variable fragment (scFv) antibodies have been generated by phage display technology, which enables the detection of HS glycosaminoglycan structures in situ (22)(23)(24). These antibodies have been used to probe HS in a variety of tissues, including kidney (22,24,25), skeletal muscle (23,26,27), spleen (28), pancreas (29), fetal lung (30), and adult lung (31), and in disease, such as metastatic malignant melanoma (32). However, their application is complicated by the fact that they are raised against heterogeneous native heparin and HS chains, and consequently the exact structures they recognize within the polysaccharide are unknown.…”

Proteoglycomics: Recent Progress and Future Challenges

“…Phage display mAbs bind GAG epitopes with higher affinity and specificity than the IgM type mAbs generated by immunization and have been used in spatial profiling of HS structural domains in kidney (Dennissen et al, 2002;Van Kuppevelt et al, 1998), uterine myometrium (Van Kuppevelt et al, 1998), developing mouse intercostal muscle , developing rat lung (Thompson et al, 2007), as well as in characterization of eight anticoagulant heparinoids (Wijnhoven et al, 2008).…”

“…Immunofluorescence microscopy imaging permits visualization of PG expression and spatial distribution in cells and tissues, and currently a number of wellcharacterized monoclonal antibodies (mAbs) are available commercially that react specifically with PG-related epitopes. Two IgM type mAbs, 10E4 and 3G10, first characterized by David et al (1992) have been used in several studies for the HSPG immunodetection (David et al, 1992;Thompson et al, 2007;Van Den Born et al, 2005;Yokoyama et al, 1999). An epitope recognized by mAb 10E4 is present in native HS and contains at least one N-sulfo group, whereas mAb 3G10 does not react with intact HS but recognizes HS or HSPGs treated with heparinase III (E.C.4.2.2.8), which generates an unsaturated uronic acid residue attached to an N-acetylglucosamine through a b 1-4 linkage (David et al, 1992).…”