ESSENTIALS OF GLYCOBIOLOGY LECTURE 4 MONOSACCHARIDE METABOLISM AND SUGAR NUCLEOTIDE TRANSPORTERS Hud Freeze

INTRODUCTION Traditional Approach--Study the enzymes and substrates in cells Holistic Approach--Add homeostasis and disease considerations Glucose is the central player, but not the only player Monosaccharides = sugars First Principles Meet the players Source of sugars: Transport into cells salvage them make them in cells Activate the sugars Deliver activated sugars to the proper location

Glc Man Gal GalNAcGlcNAc Fuc GlcA Xyl Sia THE SUGAR LEAGUE Fru-6-P

THREE WAYS TO ACTIVATE A SUGAR

Activated Sugar Donors

MONOSACCHARIDE TRANSPORTERS

Isoform Main Tissue Localization Functional Characteristics (Transport) GLUT1 Erythrocytes, brain, ubiquitous Glucose GLUT2 Liver, pancreas, intestine, kidney Glucose (low affinity); Fructose GLUT3Brain Glucose (high affinity) GLUT4 Heart, muscle, adipose tissue, brain Glucose (high affinity) GLUT5 Intestine, testes, kidney Fructose; Glucose (very low affinity) GLUT6 Brain, spleen, leucocytes Glucose GLUT7n.d.n.d. GLUT8 Testes, brain and other tissues Glucose GLUT9 Liver, kidney n.d. GLUT10 Liver, pancreas Glucose GLUT11 * Heart, muscle Glucose (low affinity); Fructose (long form) GLUT12 Heart, prostate, muscle, small intestine, adipose tissue n.d. GLUT13 Testes specific n.d. GLUT14Brain H + –myo–inositol * GLUT11 occurs in two splice variants: a short from (low-affinity glucose transport and a long form (which may be a fructose transporter).

G P NH 2 N GRK GR E RG w PESPRYL ERVGRR PETKG E Y Y COOH G G Facilitated Diffusion Transporter Topology and Signature Motifs

GLUT1 GLUT2 GLUT4 GLUT3 GLUT14 GLUT8 GLUT6 GLUT10 GLUT12 GLUT5 GLUT9 GLUT11 HMIT fructose glucose/fructose H + -myo-inositol erythrocytes, brain liver, islets brain, testis, ovary muscle, fat, heart Intestine, testis, kidney spleen, leukocytes, brain blastocysts, brain, testis liver, kidney liver, pancreas heart, muscle heart, prostate brain testis Human Hexose Facilitated Diffusion Transporters SPECIFICITY NAME LOCALIZATION

OVERVIEW OF SUGAR METABOLISM IN CELLS Remember: 1. Glucose is central 2. Pathways not = in all cells 3. Map is 2-D, not 3-D

Galactosemia is caused by a Failure in Gal-1-P:UDP-Glc Uridyl transferase

GALACTOSE ACTIVATION USES ALL THE OPTIONS

GALACTOSE METABOLISM

UDP-Glc + Gal-1-P Glc-1-P + UDP-Gal X Galactosemia Inability to metabolize Galactose--potentially lethal Treatment: Galactose restricted diet Even with proper diet: patients have dyspraxic speech, ovarian failure, poor growth, neurological impairment Knockout mouse does not have disease phenotype! Other genes important

Mannose supplements can treat a human disorder: Fru-6-P-->Man-6-P

Oral fucose supplements used to treat a human genetic disorder

UDP-GlcNAc epimerase/kinase Defective in two human diseases

Fru-6-P is the only freely soluble glycolytic intermediate Are the enzymes and substrates in the cytosol really soluble?

If all the sugars can be inter-converted, how can you follow just one?

Remains as [2- 3 H]Mannose Remains as [4- 3 H]Galactose TRICKS OF THE TRADE

?s These are the Major Leaguers, how about life in the Minors?

BIOSYNTHESIS OF UNUSUAL SUGARS KDN--2-keto-3-deoxy-D-glycero-D-galactonononic acid a sialic acid analog found mostly in fish eggs Man--->Man-6-P---> KDN---> CMP-KDN + + PEPCTP Glycoproteins, glycolipids Galactofuranose--Galf, found in bacteria and lower eukaryotes pf UDP-Galp---->UDP-Galf----> Glycoconjugates mutase Galactofuranosyl transferases

From: Schenk et al Glycobiol 11, 61R, 2001 SYNTHESIS OF DOLICHYL PHOSPHATE

All of the precursors are made in the cytoplasm or nucleus-- BUT, most glycoconjugates are made in the Golgi or ER

TRANSPORTERS IN THE GOLGI

TRANSPORTERS IN THE ENDOPLASMIC RETICULUM ?

HOW IT WORKS

TRANSPORTERS: A FAMILY IN LOVE WITH ITS MEMBRANES

DONORS AND CARRIERS FOR GLYCAN MODIFICATIONS MODIFICATIONPRECURSORTRANSPORTER Phosphate ATPYes Sulfate PAPSYes Methyl AdoMet? Acetyl Acetyl-CoAYes Pyruvate PEP? Acyl Acyl-CoA (?)? Succinyl Succinyl-CoA (?)?

SUMMARY AND TAKE HOME MESSAGES Glucose can be made into all sugars (monosaccharides) All sugars require activation and most require transport of the activated forms Transporters deliver the sugars to cells Salvaged and imported sugars can contribute to glycoconjugate synthesis The relative contributions of each source may be cell/tissue specific Some disorders in sugar metabolism can be treated with dietary modifications Hard to know specificity of sugar nucleotide and monosaccharide transporters Sugar is good for you---mostly