Alteration of Polysaccharide Size Distribution of a Vertebrate Hyaluronan Synthase by Mutation

Paul L Deangelis,Philip E Pummill

doi:10.1074/jbc.m301097200

Abstract

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has long been known to play structural roles in vertebrates. Recently, it has become increasingly obvious that this linear polysaccharide has many more uses than simply scaffolding or space filler. HA has been found to be involved in development, cell signaling, cell motility, and metastasis. These roles are often dictated by the length of the HA polymer, which can vary from a few to about 10,000 sugar residues in length. Three distinct isoforms of HA synthase exist in mammals. It has been shown previously by others that each isoform produces HA that differs in size distribution, but the regulatory mechanism is not yet known. Mutations have been described that alter the size distribution of the HA produced by the streptococcal HA synthases. We show that by mutating one particular amino acid residue of a vertebrate HA synthase, depending on the introduced side chain, the size of HA produced can be either reduced or increased. We postulate that several cysteine residues and a serine residue may be involved in binding directly or indirectly to the nascent HA chain. These data support the theory that the relative strength of the interaction between the catalyst and the polymer may be a major factor in HA size control.

Highlights

HA1 is a glycosaminoglycan composed of repeats of the alternating disaccharide [34]-␤-D-GlcUA[133]-␤-D-GlcNAc[13]
HA in the culture medium from mammalian cells transfected with HAS1, 2, or 3 showed differences; HAS1 and 3 appeared to produce HA with more equivalent size distributions in vivo but still smaller than HAS2
Another group showed that the size distribution of HA products formed by xlHAS1 and xlHAS2 differed substantially when examined in vitro [51]

Summary

EXPERIMENTAL PROCEDURES

Production of Recombinant xlHAS1 Wild Type, Cysteine Mutants, and Ser Mutant Enzymes—All of the reagents were from Sigma or Fisher unless noted otherwise. The membranes (40 –1400 ␮g of total protein) were incubated with 0.3– 0.6 mM UDP[14C]GlcUA (0.1– 0.2 ␮Ci) and 1.2–2.4 mM unlabeled UDP-GlcNAc in 50 mM Tris, pH 7.5, and 20 mM MgCl2 at 30 °C for various times. The sizes of HA polymers were analyzed by gel filtration chromatography on a Phenomenex PolySep-GFC-P 5000 or 6000 column (300 ϫ 7.8 mm) eluted with 0.2 M sodium nitrate at 0.6 ml/min on a Waters 600E system. The columns were standardized with various size dextrans (580, 145, 50, and 20 kDa) or, more appropriately, MANT-labeled HA with average molecular masses of 1300, 600, and 80 kDa (determined by MALLS). Each radiolabeled sample was spiked with an internal fluorescent dextran standard; elution times were reproducible to within 0.1 min. Protein A-alkaline phosphatase detection with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium was used to visualize the immunoreactive bands

RESULTS AND DISCUSSION

WT ϩϩ

Long incubation