Role of a Single Amino Acid in the Evolution of Glycans of Invertebrates and Vertebrates

Structures of glycoconjugate N-glycans and glycolipids of invertebrates show significant differences from those of vertebrates. These differences are due largely to the vertebrate β1,4-galactosyltransferase-1 (β4Gal-T1), which is found as a β1,4-N-acetylgalactosaminyltransferase (β4GalNAc-T1) in invertebrates. Mutation of Tyr285 to Ile or Leu in human β4Gal-T1 converts the enzyme into an equally efficient β4GalNAc-T1. A comparison of all the human β4Gal-T1 ortholog enzymes shows that this Tyr285 residue in human β4Gal-T1 is conserved either as Tyr or Phe in all vertebrate enzymes, while in all invertebrate enzymes it is conserved as an Ile or Leu. We find that mutation of the corresponding Ile residue to Tyr in Drosophila β4GalNAc-T1 converts the enzyme to a β4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold. Furthermore, we find that, similar to the vertebrate/mammalian β4Gal-T1 enzymes, the wild-type Drosophila β4GalNAc-T1 enzyme binds to a mammary gland-specific protein, α-lactalbumin (α-LA). Thus, it would seem that, during the evolution of vertebrates from invertebrates over 500 million years ago, β4Gal-T1 appeared as a result of the single amino acid substitution of Tyr or Phe for Leu or Ile in the invertebrate β4GalNAc-T1. Subsequently, the pre-existing α-LA-binding site was utilized during mammalian evolution to synthesize lactose in the mammary gland during lactation.