Numerous studies have been conducted of KS in the developing avian cornea since the early investigations of Anseth
10 in 1961 and Hart
11 in 1976. The extent to which poly-N-lactosamine chains of KS are substituted with sulfate groups is likely to be critical in terms of the molecular interactions of KS PGs. Sulfation of KS would be expected to directly affect the molecular polarity of KS PGs, contributing to their water-binding properties and their influence on tissue hydration
20 and collagen interfibrillar spacing. Antibodies recognizing specific sulfation motifs on KS chains enable precise mapping by immunomicroscopy of the tissue location and extent of sulfation of KS PGs with a resolution down to the level of single collagen fibrils. Antibody 5D4 immunoreactivity has a requirement for linear sequences of
N-acetyllactosamine that are minimally pentasulfated and that we normally regard as high-sulfated KS epitope, whereas antibody 1B4 recognizes lesser sulfated oligosaccharide units.
17 18 In the present study, immunodetection of low-sulfated KS epitope indicated low, but gradually increasing, levels throughout chick corneal development, whereas the high-sulfated epitope, identified by antibody 5D4, consistently appeared more abundant. 5D4 labeling showed that KS chains in embryonic chick corneal stroma express high-sulfated epitopes from an early stage, as reported previously.
12 13 More intense labeling for high- and low-sulfated epitopes was present in anterior, compared with posterior, stroma at days 12 and 14, extending to uniform distribution by day 18. This observation differs from earlier immunohistochemical studies in which increased posterior
12 or uniform stromal localization of KS was reported.
13 However, increased KS biosynthesis in the anterior stroma might be expected because this location coincides with the region of greatest cell density and collagen deposition at these developmental stages and may also be subject to low intracorneal oxygen tension, which some evidence suggests is a factor that predisposes toward KS biosynthesis.
21 22 Weaker staining with antibody 5D4, as we observed in the posterior stroma, has previously been interpreted as undersulfation of KS,
13 but this is perhaps less likely than the possibility that KS chains have not yet attained full length at this developmental stage, bearing in mind that current evidence suggests that sulfation of specific residues on KS poly-
N-acetyllactosamine is linked to elongation of the KS chain.
23 24 25