New approaches for mapping the recognition sites in protein–GAG interactions: a study of GAG binding by hepatocyte growth factor/scatter factor
Authors: Deakin Jon A. , Lietha Daniel , Gherardi Ermanno , Gallagher John T. , Lyon Malcolm 
Published: August 2004

Introduction  The minimal size and structure of GAG that binds to a protein, as well as the site of GAG interaction on the protein surface, are crucial recognition features for understanding, and potentially modulating, protein–GAG interactions. Methods that are easily and generally applicable for investigating such interactions, irrespective of the GAG species involved, are very limited. We have been developing such methodologies using the example of hepatocyte growth factor/scatter factor (HGF/SF). We have previously shown HGF/SF to have an unusual GAG‐binding specificity, in that it binds with high affinity to, and is activated by, both heparan and dermatan sulfates. There are two naturally occurring, highly truncated variants of HGF/SF. These comprise only the N‐terminus and either the first one (NK1) or two (NK2) of the four Kringle domains of the α‐chain of HGF/SF, and without a β‐chain. Some controversy presently exists as to whether GAG‐binding in HGF/SF requires the N‐terminus alone, or additionally the second Kringle domain.

Materials and methods  We have developed a protocol for analysing protein–GAG interactions utilizing fluorescently tagged GAG oligosaccharides run in a protein gel mobility shift assay under native associative conditions. The activity of oligosaccharides has been assessed by their ability to stimulate HGF/SF‐mediated activation of signals downstream of the Met receptor in sulfated GAG‐deficient CHO pgsA‐745 cells.

Results  HS tetrasaccharides and DS hexasaccharides are the minimal binding and activatory species for HGF/SF. The basis for this size difference between HS and DS species is not presently known. However, their ability to efficiently compete with each other does indicate a shared binding site. The three proteins HGF/SF, NK1 and NK2 all display identical GAG‐binding specificities and minimal oligosaccharide‐binding sizes. This suggests that NK1, the smallest variant, contains the entire minimal GAG‐binding functionality of full‐length HGF/SF, and thus the second Kringle domain is not required. Identification of NK1 and tetrasaccharides as the minimal binding partners has allowed us to directly target the binding site on NK1. Fluorescently tagged tetrasaccharides have been crosslinked into the binding site on NK1 using the zero‐length crosslinking methodology. After tryptic digestion, the specific fluorescently tagged peptides can be recovered for subsequent sequencing to locate the GAG‐binding site within the known amino acid sequence of the protein.

Discussion  Methods have been developed which can help to identify the respective domains on both GAG and protein involved in GAG‐protein recognition. These are relatively easy approaches, applicable to physiological solution conditions and having the advantage of being widely applicable to many protein interactions involving any uronate‐containing GAGs. In addition to HGF/SF, the gel mobility shift assay has also been applied by us to a variety of other diverse proteins.