Presentation on theme: "Proteoglycans and Glycosaminoglycans Natasha E. Zachara Ph.D. The Department of Biological Chemistry."— Presentation transcript:
Proteoglycans and Glycosaminoglycans Natasha E. Zachara Ph.D. The Department of Biological Chemistry
Key to Glycan Structures Both editions of Essentials of Glycobiology are freely available online. Focus on Chapters 15, 16, 35, 37 and 44. Other articles used in the preparation of this lecture are listed at the bottom of the relevant slide. Chapter 1, Figure 5 Essentials of Glycobiology Second Edition
Classes of Glycoproteins: O -linked glycosylation, describes carbohydrates bound to the protein backbone through hydroxyl residues, such as those in serine (Ser), threonine (Thr), tyrosine (Tyr), hydroxylysine (hLys), and hydroxyproline (hPro). – Mucin-like – Phosphoglycosylation – O-GlcNAc and N -linked glycosylation, typically refers to the amide bond formed between GlcNAc and Asn in the -conformation. C -mannosylation, is the attachment of an a-Man to carbon-2 of the indole ring of tryptophan (Trp). Glycosylphosphatidyl inositol (GPI)-anchors, GPI-anchors link the terminal residue of a protein through phosphoethanolamine and a carbohydrate core to a lipid moiety which anchors the protein in the lipid bilayer. Proteoglycans: Proteins that are glycosylated by one or more glycosaminoglycan chains.
What are: Proteoglycans: Any protein with one or more covalently attached glycosaminoglycan chains. Glycosaminoglycans (GAGs): Polysaccharide side chains of proteoglycans or free complex polysaccharides composed of linear disaccharide repeating units, each composed of a hexosamine and a hexose or a hexuronic acid. Glycosaminoglycans include: Heparin, Heparan sulfate, Chondroitin sulfate, Dermatan sulfate, and Hyaluronan. Hyaluronan or hyaluronic acid: A glycosaminoglycan defined by the disaccharide unit (GlcNAcβ1–4GlcAβ 1–3) n that is neither sulfated nor covalently linked to protein. Hyaluronan is NOT typically added to proteins, which is unlike other glycosaminoglycans.
Introduction: Proteoglycans Also known as mucopolysaccharides: – First studies proteoglycans included an anticoagulant (heparin) from liver and chondromucoid from the cartilage The glycosaminoglycan chains of proteoglycans are linear and can be up to 80 sugars long: – >1 GAG can be linked to a protein (for example aggrecan, >100 GAGs) – These can form gels, similar to mucins Present in virtually all extracellular matrices: – Interact with fibrous proteins that provide tensile strength and elasticity, as well as and adhesive glycoproteins to provide a hydrated gel that resists compressive forces Cartilage – proteoglycan From:
Proteoglycans can be membrane bound or secreted Chapter 16, Figure 2 Essentials of Glycobiology Second Edition
Proteoglycan Components Proteoglycans display structural variation: – There are a large number of core proteins (20-450kDa) – These can be modified by one or more types of glycosaminoglycan – There is variation in chain length, and stoichiometry – Proteoglycans can also be modified by O-linked and N-linked glycans, as well as GPI anchors – The GAG component can be further modified by sulfation, phosphorylation, fucosylation, and sialylation
GAG can be Classed into 6 Major Types, of Which Five are Found Covalently Bound to Proteins ① Heparin ② Heparan sulfate ③ Chondroitin sulfate ④ Dermatan sulfate ⑤ Keratan sulfate ⑥ Hyaluronan (not typically added to proteins) What do these all have in common: ➤ Repeating units of hexosamine and a hexose or a hexuronic acid n n
Glycosaminoglycans consist of repeating disaccharide units Chapter 16, Figure 3 Essentials of Glycobiology Second Edition GlcNAc GalNAc Galactose Glucuronic acid Iduronic acid
Heparin versus Heparan Sulfate HS is made by virtually all cell types, whereas Heparin is produced in Mast cells Chapter 16, Table 7 Essentials of Glycobiology
Chapter 3 Figure 1 Essentials of Glycobiology Biosynthesis of Proteoglycans
Keratan Sulfate Keratan Sulfate I: N-glycan core – ~50 disaccharides, 20–25 kD – a mixture of non-sulfated, mono- sulfated (Gal-GlcNAc6S), and di- sulfated (Gal6S-GlcNAc6S) disaccharide units. – Sulfation of a terminal galactose blocks chain elongation Keratan Sulfate II: O-glycan care (Ser/Thr) Chapter 16, Figure 4 Essentials of Glycobiology Second Edition KS consists of a polysulfated poly-N- acetyllactosamine structure identical to that found on conventional glycoproteins and mucins
The Synthesis of Chondroitin Sulfate, Dermatan Sulfate, Heparin and Heparan Sulfate is Initiated by the Same Enzyme in the ER Chapter 16, Figure 3 Essentials of Glycobiology Second Edition GlcNAc GalNAc Galactose Glucuronic acid Iduronic acid
The biosynthesis of CS and HS is initiated by the formation of a linkage region tetrasaccharide Chapter 16, Figure 5 Essentials of Glycobiology Second Edition A glycine residue is often found on the carboxy terminal side of the serine attachment site. 2 Acidic residues nearby the glycosylated serine are also common features. But, there is no strict consensus motif.. These enzymes are important control points because they ultimately regulate the type of glycosaminoglycan chain assembled. There is some evidence that the surrounding amino acid dictates recognition.
Biosynthesis of chondroitin sulfate/dermatan sulfate Chapter 16, Figure 6 Essentials of Glycobiology Second Edition
Biosynthesis of heparan sulfate Chapter 16, Figure 7 Essentials of Glycobiology Second Edition Extl3 recognizes a linear acidic sequence in proteins.
Sulfation provides a finger print which define different biological functions In contrast to chondroitin chains, which tend to have long tracts of fully modified disaccharides, the modification reactions in HS biosynthesis occur in clusters along the chain, with regions devoid of sulfate separating the modified tracts. This leads to NA domains, NA/NS domains, and NS domains. Chapter 16, Figure 8 Essentials of Glycobiology Second Edition
Functions of Proteoglycans Interstitial Proteoglycans: – Bind water and form hydrated matrices – Fills the space between cells – Can absorb compressive loads Help organize the basement membranes, providing a scaffold for epithelial cell migration, proliferation, and differentiation Can regulate the permeability of specialized BM. Can bind cytokines, chemokines and protect them from proteolysis, facilitating the formation of morphogen gradients Can act as co-receptors for tyrosine kinases.
Interstitial Proteoglycans: Aggrecan Aggrecan family: – Aggrecan family of proteoglycans consists of aggrecan, versican, brevican, and neurocan – The interstitial proteoglycans appear to be unique to vertebrates – Characterized by a N-terminal Hyaluronan binding domain – C-terminal lectin domain – Generally modified by Chondroitin sulfate, and occasionally keratan sulfate
Aggrecan Aggrecan is a critical component for cartilage structure and the function of joints; Can be modified by >100 GAG chains. Forms a gel with the ability to resist compressive loads Functionally, the G1 domain interacts with hyaluronan and link protein, forming stable ternary complexes in the extracellular matrix Aggrecan plays an important role in mediating chondrocyte-chondrocyte and chondrocyte-matrix interactions through its ability to bind hyaluronan
Interstitial Proteoglycans: SLRPs Small leucine rich proteoglycans (SLRPs) – 9 members – Modified by Dermatan sulfate, chondroitin sulfate, keratan sulfate – Interacts with collagen Stabilizes collagen in tendons – In the eye: Decorin, Lumican, Keratocan, Mimecan Relatively uniform size with only 1-2 Gags maintain the even spacing of type I collagen fibrils in the cornea Fit between collagen fibers in the eye, maintain the spacing and thus transparency Mutations in Keratan sulfate synthesis lead to macular corneal dystrophy
Membrane Bound Proteoglycans For example Syndecan – Single membrane pass Facilitate cellular interactions Binding of Syndecan to HA can induce oligomerization; and recruitment of Kinases, PDZ-domain proteins and cytoskeletal components to the cytoplasmic domain Glypican – Characterized by GPI- Anchors – An N-terminal globular domain – Only carry HS – Bind numerous factors essential for morphogenesis and development GPC3: loss of GPC3 results in Simpson-Golabi-Behmel syndrome, characterized as an overgrowth disorder – suggesting that GPC3 acts to inhibit cell growth.
Binding of growth factors to proteoglycans can induce oligomerization and thus signaling Trends in Cell Biology, Volume 19, Issue 3, 2009,
Many functions of proteoglycans are mediated by proteins which bind GAGs ClassExamplePhysiological/pathophysiologic al effect Enzymesglycosaminoglycan biosynthetic enzymes, thrombin and coagulation factors (proteases), complement proteins (esterases), extracellular superoxide dismutase, topoisomerase multiple Enzyme inhibitorsantithrombin III, heparin cofactor II, secretory leukocyte proteinase inhibitor, C1-esterase inhibitorheparin coagulation, inflammation, complement regulation Cell adhesion proteins P-selectin, L-selectin, some integrinsselectin cell adhesion, inflammation, metastasis Extracellular matrixExtracellular matrix proteins laminin, fibronectin, collagens, thrombospondin, vitronectin, tenascincell adhesion, matrix organization Chemokinesplatelet factor IV, γ-interferon, interleukinschemotaxis, signaling, inflammation Growth factorsfibroblast growth factors, hepatocyte growth factor, vascular endothelial growth factor, insulin-like growth factor–binding proteins, TGF-β-binding proteins mitogenesis, cell migration Morphogenshedgehogs, TGF-β family memberscell specification, tissue differentiation, development Tyrosine-kinase growth factor receptors fibroblast growth factor receptors, vascular endothelium growth factor receptor receptor mitogenesis Lipid-binding proteins apolipoproteins E and B, lipoprotein lipase, hepatic lipase, annexinslipid metabolism, cell membrane functions Adaptation, Table 35.1 Essentials in Glycobiology
Proteoglycans and Signaling Gradients https://www.neb.com/sitecore/content/nebsg/home/applications/glycobiology/depolymerization-of-heparin-hs
The FGF Receptor & Heparin >22 growth factors bind heparin FGF2 has a very high affinity for heparin (Kd ~ 10 −9 M) FGF2 has potent mitogenic activity in cells Heparin promotes the mitogenic response by promoting dimerization of the FGF receptor Technically a pentasaccharide is required for binding, although it’s only longer oligomers that trigger a biological response
Congenital Exostosis Defects in the formation of heparan sulfate (HS) cause hereditary multiple exostosis (HME)heparan sulfate an autosomal dominant disease with a prevalence of about 1:50,000 It is caused by mutations in two genes EXT1 and EXT2, which are involved in HS synthesis. HME patients have bony outgrowths, usually at the growth plates of the long bones. HME mutations occur in EXT1 (60–70%) and EXT2 (30–40%). However, the partial loss of one allele of either gene appears sufficient to cause HME. This means that haplo-insufficiency decreases the amount of HS and that EXT activity is rate limiting for HS biosynthesis. The mechanism of HME pathology is likely rooted in a disruption of the normal distribution of HS-binding growth factors, which include FGF and morphogens such as hedgehog, Wnt, and members of the TGF-β family.
How does disrupting signaling alter neuronal development? Heparan sulfate was eliminated from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Mutant mice recapitulated a range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization. From these and other experiments they concluded that AMPA receptor-mediated synaptic transmission is compromised in the absence of HS, presumably because of the reduced synaptic expression of AMPA receptors. Proc Natl Acad Sci U S A Mar 27;109(13): doi: /pnas Epub 2012 Mar 12. PMID:
Proteoglycans and Signaling Gradients Mutations in the Heparin Sulfate biosynthetic machinery lead to defects in Wnt signaling. Cold Spring Harb Perspect Biol.Cold Spring Harb Perspect Biol Sep;1(3):a PMID:
Proteoglycans and Signaling Gradients Mutations in the Heparin Sulfate biosynthetic machinery lead to defects in Wnt signaling; Wnt can not diffuse across cells without two glypicans, Dally and Dlp. There is also evidence suggesting that Dally presents Wnt to the dFz2 signaling receptor, promoting an activation of signaling. Bornemann D J et al. Development 2004;131:
Cells Secrete Heparanase to Release Growth Factors Chapter 16, Figure 10 Essentials of Glycobiology Second Edition Extracellular heparanase can cleave HS chains at restricted sites, resulting in release of growth factors or chemokines immobilized on HS proteoglycans at cell surfaces or in the ECM. The sulfation of Heparan Sulfate can also be modulated at the cell surface, resulting in an altered response of cells to growth factors and morphogens.
Anti-Thrombin & Heparin/HS Interactions heparin is used clinically as an anticoagulant
Anti-Thrombin & Heparin/HS Interactions heparin is used clinically as an anticoagulant Anti-thrombin is a protease inhibitor of the Serpin family Heparin activates anti-thrombin – Change in conformation which results in a 1000x enhancement of the rate of inactivation of thrombin and factor Xa – Heparin also acts to bring thrombin and anti-thrombin together Chapter 35, Figure 2 Essentials of Glycobiology Second Edition
Degradation of GAGs Chapter 16, Figure 9 Essentials of Glycobiology Second Edition
Mucopolysaccharidosis Defects in GAG catabolism A group of rare inherited lysosomal storage disorders – Characterized by: abnormalities in multiple organs Reduced life expectancy Heterogeneous and progressive – MPS I, II, and IV can be treated with enzyme replacement therapy Muenzer (2011), Rheumatology, 50: V4-V12
MPS 1 There are three major types of MPS 1, which differ in severity – Hurler syndrome – described in 1919 – Scheie syndrome – described in 1962 – Hurker-Scheie – an intermediate phenotype - was discovered later Caused by defects in -L-iduronidase – which affects the degradation of dermatan sulfate and Heparan sulfate Chapter 41, Figures 3 & 4 Essentials of Glycobiology Second Edition
The Genetic Defect Autosomal recessively inherited There are more than 100 different alleles of the -L-iduronidase gene that cause all three forms of MPS I; – Thus, the designation of MPS type is arbitrary – In the mildest form, patients may remain undiagnosed for years – 653 amino acid protein, encoded from a gene on 4p16.3 MPS1 mutations include: – Introduction of premature stop codons (W402X, Q70X) These are the most common mutations (70%), >13 additional mutations that introduce stop codons No detectable protein – D349N, E182A and E299A Lead to reduced protein levels Alter protein folding, leading to export and degradation from the ER – A75T does not alter protein levels, but ablates enzyme activity
Treatments ➤ Enzyme replacement therapy (ERT) ➤ Laronidase (Genzyme), approved since 2003 ➤ Infusions every other week (1-4h) ➤ Clinical outcomes: Reduced urinary GAGs, reduced liver volume, improved vital capacity, improved walking tests ➤ Risks: anaphylaxis ➤ Haematopoitic stem cell transplantation (HSCT) ➤ Bone marrow transplantation most effective in young patients ➤ Best treatment for more severe MPS 1 ➤ Improves median age from 6.8 years to beyond 20 years ➤ Maintains cognition, but musculoskeletal disease continues to progress along with reduced vision and poor growth. ➤ These are not ideal, in part as secondary damage from GAG accumulation is often irreversible; ➤ Early diagnosis improves treatment outcomes.
What are: Proteoglycans: Any protein with one or more covalently attached glycosaminoglycan chains. Glycosaminoglycans: Polysaccharide side chains of proteoglycans or free complex polysaccharides composed of linear disaccharide repeating units, each composed of a hexosamine and a hexose or a hexuronic acid. Glycosaminoglycans include: Heparin, Heparan sulfate, Chondroitin sulfate, Dermatan sulfate, and Hyaluronan. Hyaluronan or hyaluronic acid: A glycosaminoglycan defined by the disaccharide unit (GlcNAcβ1–4GlcAβ 1–3) n that is n either sulfated nor covalently linked to protein.
What is Hyaluronan? A high molecular weight glycosaminoglycan; Purified from bovine vitreous humor in the 1930’s by Myer and Palmer (1934); hyaluronic acid: from hyaloid [meaning vitreous] and uronic acid (as it contained uronic acid and an amino sugar); HA is not attached to proteins, is not sulfated or fucosylated, and contains no Iduronic acid; Largest polysaccharide in vertebrates. Chapter 15, Figure 1 Essentials of Glycobiology Second Edition In the beta configuration of the bulky groups (hydroxyls, carboxylate moieties, anomeric carbon) are in the sterically favorable equatorial positions. Thus, the structure of the HA disaccharide is energetically favorable.
HA arose in evolution at the same time as the notochord Made by virtually all cells from vertebrates, and its expression correlates with tissue expansion and cell motility; Made by animal cells, but not Drosophila melanogaster and Caenorhabditis elegans; Sometimes found in the capsule of some strains of Streptococci. This is quite likely pirated enzymatic machinery (from vertebrate hosts).
Hyaluronan has a large hydrodynamic volume Can be up to 10 4 disaccharides long, ~4 × 10 6 Daltons; Up to10 M in length, thus stretching half way around the cell; Polyanionic; At 3-5mg/ml HA occupies all of the solvent; Thus, it can filter out large molecules while allowing small molecules to pass; HA is stiffened by a combination of the chemical structure of the disaccharide, internal hydrogen bonds, and interactions with solvent; a hyaluronan molecule assumes an expanded random coil structure in physiological solutions; Has swelling pressure and high viscosity, and is essential for distributing load in the joints. ml
Hyaluronan has a large hydrodynamic volume In some tissues HA can be a major constituent: – in the vitreous of the human eye HA is mg/g wet weight); – in synovial joint fluid (3-4 mg/ml). The largest amount of hyaluronan resides in the skin – (7-8 g per average adult human; – ~50% of the total in the body. https://askanesthetician.files.wordpress.com/2011/01/hyaluronic-acid1.jpg
Hyaluronan Biosynthesis HAS or Hyaluronan synthetase is the bifunctional glycosyltransferase; In mammals there are three homologs: – HAS2 appears to be the most essential; – Unlike other forms of protein glycosylation, extension occurs at what might be considered the reducing terminal end; HA is exuded into the ECM as part of the biosynthetic process; 5-6 possible trans-membrane domains; Metabolic studies have shown that the half life of a hyaluronan molecule in cartilage is normally 2-3 weeks. Chapter 15, Figure 2 Essentials of Glycobiology Second Edition
HA Degradation ~5 grams of HA is turned over each day, ~1/3 of that in the human body; The endothelial cells of the lymph nodes and liver sinusoids remove HA via specific receptors – LYVE-1 (a homolog of CD44) – HARE (hyaluronan receptor for endocytosis); Large molecules of HA are clipped by a GPI-anchored hyaluronidase, most likely Hyal2; HA is ultimately degraded in the lysosome. Chapter 16, Figure 9 Essentials of Glycobiology Second Edition
HA Function HA can play a structural role: it has swelling pressure and high viscosity, and is essential for distributing load in the joints. HA increases levels of tissue hydration, which can facilitate movement of cells through tissues. Promotes the assembly of of extracellular matrices through specific interactions with other macromolecules; HA interacts with several types of cell-surface receptors, especially CD44 and RHAMM. Thus, HA plays roles in development, tissue organization, cell proliferation, diabetes, stress and inflammation; – HA binding proteins; – ECM Assembly; – Ovulation; – Cell Signaling; – Fertilization.
Many of Hyaluronan’s Functions are Dependent on HA binding Proteins Chapter 15, Figure 3 Essentials of Glycobiology Second Edition Link domains are characterized are ~100 amino acids in length and are similar to C-type lectins. These interactions are calcium independent. There are alternative domains, many of which have a or a BX 7 B motif.
Link Proteins: Linking Proteoglycans to Hyaluronan Chapter 16, Figure 1 Essentials of Glycobiology Second Edition Hyaluronan often forms a scaffold for the binding of other proteoglycans, such as the connective tissues surrounding smooth muscle cells in the aorta and fibroblasts in the dermis of skin; Aggrecan is one protein with a LINK domain – and deletion of the Link domain results in mice with defects in cartilage and bone formation.
In some situations HA can be covalently linked to a protein: Inter- Trypsin-inhibitor Inter-αTrypsin Inhibitor (IαI) is found in serum at 0.5mg/ml; IαI is a trypsin inhibitor, but a poor inhibitor of physiologically relevant proteases such as elastase and kallikrein; Contains two heavy chains linked to chondroitin sulfate through an ester linkage to GlcNAc. The chondroitin sulfate in turn modifies bikunin. The Inter-αTrypsin inhibitor can be transferred from Chondroitin sulfate to HA generating the Serum-derived Hyaluronan-Associated Protein (SHAP)
Hyaluronan and Ovulation The oocyte is surrounded by closely adherant cumulus cells to form the compact COC or cumulus cell-oocyte complex; Gonadotropin: – Resumption of meiosis by the oocyte; – Permeabilization of the follicle to large serum proteins including the I I, which is essential for expansion of the COC; – Synthesis of a HA (upregulation of has2) rich ECM, which in part promotes expansion of the COC. HA reaches a concentration of ~0.5 mg/ml); – Crosslinking of the HA, probably via I I is essential for expansion; – HA promotes detachment of the oocytes from the follicular wall; – HA also appears to promote the capture of the release oocyte by the oviduct.
HA, Cell Adhesion, and Locomotion HA is expressed abundantly during morphogenesis and in both physiological and pathological invasive processes; Has2-null mouse is embryonic lethal phenotype; Has2-null embryonic heart do not synthesize hyaluronan or undergo endothelial-mesenchymal transformation and migration; Co-culturing Has2 wild-type and null embryonic cells, or adding hyaluronan to the culture rescues the phenotype. Exp Clin Cardiol.Exp Clin Cardiol Spring;6(1):4-10. PMID: Conditioned mediaCo-culture Hyaluronan Boiled Hyaluronan
CD44, a HA binding protein, has altered expression in many tumors CD44 is a transmembrane receptor expressed by many cell types. CD44 is heavily glycosylated and can be subject to differential mRNA splicing. Binds to hyaluronan, and the interaction can mediate leukocyte rolling and extravasation; Changes in CD44 expression are associated with a wide variety of tumors and the metastatic spread of cancer; When hyaluronan binds to CD44, this promotes clustering of CD44, and the cytoplasmic tail interacts with regulatory and adaptor molecules, such as SRC kinases, RHO GTPases, VAV2, GAB1, and ankyrin and ezrin. Hyaluronan binding to RHAMM also transduces signals that influence growth and motility; SRC, FAK, ERK, and PKC. Chapter 37, Figure 6 Essentials of Glycobiology Second Edition
Hyaluronan Oligomers May Antagonize CD44 Based Survival Signaling Treatment with short oligomers of HA sensitized tumor cells to chemotherapeutic drugs, and inhibited survival signaling. J Biol Chem.J Biol Chem Jul 11;278(28): Epub 2003 May PMID:
Conclusions Proteoglycans are proteins modified by glycosaminoglycan chains; HA is a GAG which is NOT typically attached to proteins; GAGs play essential roles in numerous cellular processes, which is in part mediated though their interactions with other proteins; Underlying the importance of HA and proteoglycans, mutations in either the synthetic machinery or the breakdown machinery have profound effects in mammalian models.