1. Glycoproteins and mucins
Richard Vytášek 2008

2. Glycoconjugate (complex saccharide)
 molecule containing one or more carbohydrate chains covalently linked to protein (glycoprotein) or lipid (glycolipid)

3. Glycoprotein protein with covalently bound oligosaccharide chain(s)
carbohydrate chains are synthetized on polypeptide chain by highly specific enzymatic reactions from nucleotide sugars (activated sugars)
carbohydrate moiety influences specific function of glycoprotein as well as its localization and rate of degradation

4. Some functions served by glycoproteins
Structural molecules – collagen, elastin, fibrin, cell walls
Lubrication and protection – mucins, mucous secretions
Imnunity – immunoglobulins, histocompatibility antigens, complement, interferon
Hormone – chorionic gonadotropin, TSH
Enzymes – hydrolases, clotting factors
Transport molecules – minerals, lipids, vitamins, hormones (e.g. steroids)
Cell attachment and recognition – cell to ECM, cell, bacteria, virus; membrane receptors
Lectins

5. Saccharides of glycoproteins Hexoses

6. Saccharides of glycoproteins Hexoses

7. Saccharides of glycoproteins N-Acetylhexosamines

8. Saccharides of glycoproteins N-Acetylneuraminic acid

9. Carbohydrate attachment to protein chain
1) N-linkage (via amide group of asparagine)

10. O-linkage (via hydroxyl groups of amino acids)
1) O-linkage via hydroxyl group of serine event.
threonine
   2) linkage Ser-Xyl (proteoglycans)
3) linkage hydroxylysine-Gal (collagens)

11. Synthesis of glycoproteins
Oligosaccharide chains are synthesized by highly specific glucosyltransferases from active sugars
Nucleotide sugars (active sugars) are formed in cytosol from corresponding nucleoside triphosphate under catalysis of pyrophosphorylases
UTP + Glc-1-phosphate UDP-Glc + pyrophosphate
The most of glycosylation reactions occur within lumen of Golgi apparatus and nucleotide sugars are transported across Golgi membrane from cytosol by carrier systems (permeases). The influx of one molecule of nucleotide sugar is balanced by efflux one molecule corresponding nucleotide (antiport systém)
UTP-glucose-1-phosphate uridylyltransferase

12. Synthesis of glycoproteins
synthetized oligosacharides can be modified by specific glycosidases and phosphatases
typical modification is phosphorylation of mannose in position 6. This phosphorylation determines that these glycoproteins are transported to lysosomes
the course of biosynthesis of N-linked a O-linked glycoproteins is different. N-linked oligosaccharides utilize a lipid carrier in the early stages of synthesis

16. Three main types of N-linked oligosaccharide chains

17. Sorting and transport of glycoproteins

18. Synthesis of O-linked glycoproteins
no dolichol-linked itermediates are involved
no specific sequence of amino acids is needed (the presence of hydroxyl group is sufficient)
the first sugar linked to hydroxyl group is usually GalNAc
the second sugar is usually Gal
the terminal sugars are often highly immunogenic
cytoplasmatic and nuclear proteins with single sugar are usualy synthetized in cytosol

19. Oligosacharide termini of the AB0 human blood group antigens

20. Special methods of glycoprotein research Glycosidases
enzymes cleaving glycosodic bond, they are specific for certain type of glycosidic bond, the cleavage of oligosaccharide chain(s) by various glycosidases is very usefull for structural studies (sequence of sugar residues, type of linkage etc.)
endoglycosidases cleave internal glycosidic linkage (e.g. endoglycosidase F cleaves the bond betwwen the first GlcNAc and amido group of asparagine)
exoglycosidases cleave the terminal sugar of oligosacharide chain (e.g. neuraminidase cleaves the terminal sialic acid)

21. Special methods of glycoprotein research Lectins
plant proteins that bind one or more specific sugar
purification of glycoproteins (affinity chromatography)
detection of glycoproteins - blotting (binding lectin to glycoprotein on nitrocellulose membrane) or probing of surfaces of cells (histological methods)
generation of mutant cell lines lacking certain enzymes of oligosacharide synthesis - selection of mammalian cell resistant to certain lectin, the resistance is usually caused by defficiency of the specific sugar in membrane glycoproteins and the absence of this sugar is caused by lack of certain enzymes involved in oligosacharide synthesis of these glycoproteins

22. Mucins and their biological role

23. Mucins complex glycoproteins synthetized in epithelial cells
components of mucus secretions covering epithelial cells in gastrointestinal, urogenital, tracheobronchial, ocular and auditory systems of all vertebrates (but they can be found in all eukaryotes)
very rich in carbohydrates (50-90% of mucin mass is composed by sugars) and saccharides are linked to protein via O-glycosidic bond
O-glycans are linked to serine/threonine in specific domain called tandem repeat
some mucins can also contain N-glycosidic oligosacharides, but they are bound only in cysteine-rich domain

24. Schematic drawing of a mucin glycoprotein depicting a MUC protein backbone and its O-glycans

25. Carbohydrate attachment to protein chain of mucins
Carbohydrate attachment to protein chain of mucins. O-linkage via hydroxyl group of serine or threonine
Ser Thr

26. Tandem repeats (TR) domain
characteristic mucin domain located centrally in polypeptide chain
constituted from by a repeated serine/threonine rich sequence
TR of each mucin is unique in sequence and size (5 – 395 AA)
TR domains are repeated (even more than 100 TRs per molecule)
some mucins have two or three different TR domains
only three human mucins (MUC,14,15,18) do not encode TR

27. Motifs in MUC proteins of some airway mucins.

28. Tandem repeat (TR) sequences of MUC proteins
Amino Acids/TR Repeating Sequence Number of TR/MUc
MUC GSTAPPAHGVTSAPDTRPAP ; 41 and are most common
MUC PTTTPITTTTTVTPTPTPTGTQT PPTTTPSPPPTSTTTL ; are most common
MUC3A 17 TTTETTSHSTPSFTSS TTTPNTTSHSTPSFTSSTIYSTVSTSTTAISSASPTSGT
MVTSTTMTPSSLSTDTPSTTPTTITYPSVGSTGFLTTATDLTSTFTVSSSSAMSKSVIPSSPSIQNTETSSLVSMTSATTPSLRPTITSTDSTLTSSLLTTFPSTYSFSSSMSASSAGTTHTETISSLPASTNTIHTTAESALAPTTTTSFTTSPTMEPPSTTVATTGTGQTTFPSSTATFLETTTLTPTTDFSTESLTTAMTSTPPITSSITPTDTMTSMRTTTSWPTATNTLSPLTSSILSSTPVPSTEVTTSHTTNTNPVSTLVTTLPITITRSTLTSETAYPSSPTSTVTESTTEITYPTTMTETSSPATSLPPTSSLVSTAETAKTPTTNL Unknown
MUC5AC 8 TTSTTSAP (124,17,34,66)
5 TTVGP/S
MUC5B ATGSTATPSSTPGTTHTPPVLTTTATTPT ,11,17,11,22

29. Classification of human mucins by MUC protein backbone structures
Membrane-tethered with TR (membrane mucins) – e.g. MUC1, MUC4
Secreted, cysteine-poor with TR (gel forming cystein-poor mucins) - e.g. MUC7
Secreted, cysteine-rich with TR - MUC2, MUC5AC
Mucins without TR

30. Biosynthesis of mucins
endoplasmatic reticulum -
synthesis of polypeptide chain
N-glycosylation in non-TR domains
secreted mucins create interchain disulfide bond - formation of dimer
Golgi complex -
O-glycosylation of TR domains
sulfation (sulfotransferases)
assembling of secreted mucins into multimers via further interchain disulfide linkages (trans lumen)
proteolysis at C-terminal region (e.g. MUC2)

31. Biosynthesis of secreted mucins
Motifs in MUC proteins of some airway mucins.
mannose sugar is added to tryptophan residues in Thrombospondin repeats

32. Storage and secretion of mucins
fully processed gel-forming mucins are stored in large secretory vesicles known as mucous or mucin granules (they occupy the majority of cytoplasm of mucous/goblet cells)
secretion of mucins can be
constitutive - mucous granules and/or small vesicles are exocytosed
regulated by various agents (cytokines, bacterial products, neurotransmitters, nucleotides etc) - exocytosis of mucous granules

33. Model of biosynthesis and secretion of mucin glycoproteins in a goblet or mucus cell

34. Major core types of mucin O-glycans.

35. Response of airway secretory cells to acute or chronic challenges

36. COPD/chronic bronchitis
COPD a complex of diseases characterized by airflow obstruction
chronic bronchitis is presence of cough and sputum on most days for at least 3 months per year in two years
central airways are chronically inflamed with increased numbers of macrophages and T-lymphocytes - increased levels of IL-1, IL-6, IL-8, TNF- - increased levels of protease (neutrophil and macrophages) and ROS (also cigarette smoke)
secretory cell hyperplasia causes mucus hypersecretion / mucin overproduction (e.g. elevated expression of mucins (MUC5B) ratio of MUC5B:MUC5A is higher)
structure and size of synthetized mucins are similar but less acid (decreased sulfation)