BIOCHEM 3.5 - PROTEOGLYCANS, GLYCOPROTEINS, AND GLYCOLIPIDS

Proteoglycans, Glycoproteins, and Glycolipids

Objectives

• Types of glycosidic bonds should be identified and the relationship of the bond to chemical and molecular behavior of the molecule should be explained
• Compare and contrast the structures of proteoglycans, glycoproteins, and glycolipids, differentiating them based on description provided
• Summarize the processes of N-linked glycosylation and O-linked glycosylation
• Compare and contrast the location, enzymes involved, and amino acids involved in N-linked versus O-linked glycosylation
• Assess the consequences of changes in the activity of enzymes associated with glycosylation and explain the role of glycosylation in human disease
• Assess a situation and link symptoms with the most likely cause, and/or determine the most appropriate testing to differentiate the condition from other conditions with similar symptoms

Patient Case

• A 7-month-old female presents with epilepsy, muscular hypotonia, speech, and developmental delay
• Seizures are the reason for the clinic visit
• Abnormal electroencephalogram (EEG) recordings were reported

Carbohydrates

• Nearly all sugars in the body are in the d-configuration
• Sugars are linked via glycosidic bonds
• Differences in sugar linkage contribute to differences in chemical behaviors
• Alpha-1-4-linked is different from beta-1-4-linked

Glycoconjugates

• This is a group of molecules with sugars covalently linked to monomers of the macromolecule
• Includes:
• Proteoglycans
• Glycoproteins
• Glycolipids

Proteoglycans

• This is a protein attached to glycosaminoglycan
• They are long unbranched sugar polymers potentially containing hundreds of monosaccharides
• They can be as much as 50-60% sugar by weight
• They are a highly diverse group with variable cores and variable lengths
• The saccharide chains have repeating disaccharide units
• Uronic acid (carbon containing the terminal hydroxyl group = oxidized) with an amino sugar
• (N-acetylglucosamine or N-acetylgalactosamine)
• They are important in connective tissues and act in the structural support to the extracellular matrix

Glycoproteins

• Have short glycan chains
• Typically have 3 to 15 sugars but can have up to 20
• Are 10-15% sugar by weight
• Highly branched oligosaccharides without a repeating unit
• Contain typical sugars:
• Amino
  • N-acetylglucosamine
  • N-acetylgalactosamine
• Neutral
  • D-galactose
  • D-mannose
  • 1-fucose
• Acidic
  • N-acetylneuraminic acid
• Generally do NOT contain uronic acids

Mucins

• Another group of O-linked Glycoproteins, secreted by epithelial cells (respiratory, GI, and genitourinary tracts)
• They have a wide range of linear and branching oligosaccharides
• Rich in proline, threonine and/or serine (PTS domain)
• Many have a structure containing negatively charged sialic acid that prevents tertiary folding (highly viscous)
• Protective barrier on epithelial surface
• Lubrication
• Facilitation of transport (i.e. food in GI)
• Also have a role and are expressed in the immune system

More on Glycoproteins

• Mainly present on plasma membrane in integral membrane proteins
• Have roles in cell-to-cell recognition
• Have roles in cell signaling
• The final structure of N-glycan chains is dependent on the enzymes of the cell that are generating the oligosaccharide rather than on the genetic sequence of the protein
• Glycosyltransferases
• Glycosidases

Glycolipids

• Sphingolipid (amphipathic, polar lipid) with covalently bound sugars
• Sphingosine (long chain amino alcohol) with long-chain fatty acid
• Have highly variable oligosaccharide chains
• Have polar sugar head attached to ceramide by glycosidic bond at the terminal hydroxyl of sphingosine
• There are 3 main types:
• Neutral (only neutral and amino sugars)
• Sulfatides (addition of sulfate)
• Gangliosides (contain sialic acids)

Glycosylation Introduction

• This is a posttranslational modification
• Mainly in cytosol, ER, Golgi, and sarcolemma membrane
• Contains Common amino acids for glycosylation
• N-linked
  • Asparagine
• O-linked
  • Hydroxyl group of Serine
  • Hydroxyl group of Threonine
  • Hydroxylysine (found only in collagens and proteins with collagenous domains; produced as a posttranslational hydroxylation of lysine)
  • Tyrosine (only in glycogenin)

Comparing N-glycan vs. O-glycan

• Common core with mannose or complex structures in N-glycans
• 3 or more sugars in linear or branched chains in O-glycans

N-linked Oligosaccharides Synthesis in ER

• Transfer of 2 GlcNAc residues to dolichyl phosphate (membrane bound lipid)
• Mannose and glucose residues are added, generating lipid-linked oligosaccharide intermediate
• Transfer of intermediate to protein located in Lumen of ER
• Each sugar is transferred via specific glycosyltransferases
• Glucose is NOT found on N-linked oligos
• Removed via glucosidases
• Additional processing in ER
• Translocation to Golgi
• Additional processing

Processing of N-glycans: Role of ER and Golgi

• Mannosidase I Uses Deoxymannojirimycin

Synthesis of O-linked Oligosaccharides in the Golgi

• Stepwise addition of sugars from sugar nucleotide donors
• Does NOT involve lipid intermediates
• GalNAc is transferred from UDP-GalNAc to serine or threonine residues on the protein via GalNAc transferase
• Forms GalNAc-serine protein (acceptor for Galactose and sialic acid)
• Galactose is added from UDP-Gal
• GalNAc-serine acts as acceptor
• Catalyzed by galactosyltransferases (more than 100 of these involved in a typical cell)
• Sialic acid is added from CMP-sialic acid
• Catalyzed via sialyltransferases

Glycosylation and Disease

• Most glycosylation disorders involve insufficient or incomplete glycosylation
• Point mutations in proteins can create novel glycosylation sites
• N-glycosylation profiles may provide insight and serve as biomarkers for Diabetes
• Several factors have been implicated in diabetes pathogenesis
• These factors therefore make ideal targets for analysis or treatment

Patient Case

• A 7-month-old female presents with epilepsy, muscular hypotonia, speech, and developmental delay
• Seizures are what brought the patient to clinic
• Congenital disorder of glycosylation (CDG)
• Asparagine-linked glycosylation 13 (ALG13) gene
• Located on X chromosome
• Early enzyme in N-linked glycosylation pathway
• Disorders are due to de novo or inherited missense variants
• Glycosyltransferase 28 domain altered thus impacting synthesis of oligosaccharides
• Full loss of function mutations have limited survival particularly in males
• As the patient ages, additional features develop including intellectual disability
• some cases reported in both males and females though all surviving patients reported in the study used for this case were female

Congenital Disorders of Glycosylation (CDG)

• 170 have been reported; classified as rare genetic disorders
• Has an impact on the biosynthesis of glycoproteins
• Have Multi-system pathology
• Severe impact on the nervous system
• These can be divided into 4 categories:
• N-linked glycosylation
• O-linked glycosylation
• Combined N- and O-linked/multiple glycosylation
• Lipid and glycosylphosphatidylinositol (GPI) anchor biosynthesis defects
• Screening is limited to ultrasound evaluation (common observations with multiple causes; i.e. hydrops fetalis)
• Serum transferrin isoelectric focusing (N-glycosylation with sialic acid deficiency)
• Serum apolipoprotein C-III isoelectric focusing (core 1 mucin-type-O-glycosylation)
• Diagnosis = Genetic Testing

CDG Example 1

• The PMM2-CDG (previously CDG type 1a)
• Symptoms appear in infancy, like failure to thrive; hypotonia; retracted/inverted nipples; strabismus
• There may also exhibit abnormal distribution of fat, underdeveloped cerebellum (frequently), and distinctive facial features including triangular face and high forehead
• Elevated liver function tests results, seizures, pericardial effusion and blood clotting disorders are also observed
• Around 20% will not survive past 1 year due to multiple organ failure
• Individuals who do survive past 1 year have Moderate intellectual disability, reduced ability to walk with some unable to walk independently and stroke-like episodes with lethargy
• Will exhibit temporary paralysis that can take several weeks to months for recovery and peripheral neuropathy
• N-linked protein glycosylation defect
• Hydrops fetalis observed in ultrasound (typically associated with reduced viability)
• Multiple serum glycoprotein abnormalities are observed via isoelectric focusing of transferrin Original Type 1 pattern = dolichol-linked glycan assembly and transfer defects in cytoplasm or ER
• Molecular diagnostic tools have changed the nomenclature to better fit the etiology
• Has pathogenic variants of phosphomannomutase 2 gene
• Deficiency in PMM2 enzyme
• Associated with synthesis of mannose

CDG Example 2

• The ALG6-CDG (previously carbohydrate-deficient glycoprotein syndrome type V)
• Symptoms appear in infancy, like failure to thrive; hypotonia; developmental delay
• They may also exhibit seizures/epilepsy; ataxia
• May be milder than PMM2-CDG (no stroke-like episodes or peripheral neuropathy)
• Now considered to be a common type of CDG
• Has N-linked glycosylation defect
• Pathogenic variants of ALG6 gene -Impacts attachment of 3 glucose molecules to the dolichol-linked mannose intermediates
• Results in hypoglycosylation of serum glycoproteins