HDM TBL 9: Glycoconjugates: structure, function, & disease

glycoconjugates are essential to

connective tissue

2 types of glycan on proteins

1. proteoglycan

2. glycoprotein

proteoglycan

• core protein, only O-linkage btwn glycan & protein

• located only in membranes & ECM

• mostly carbohydrate & are structural

• Glycoaminoglycan chains [GAG]

glycoprotein

• comprised of larger core protein & smaller, discrete glycans

• linkages can be N- &/or O-linkage btwn protein & glycan

  • N-linked is distinct structure to Asn

  • O-linked can be distinct or variable in size to Ser/Thr

• tend to have functions like antibody binding

• mostly protein like, have functions [ex. IgG]

*I-cell disease is clinical connect

proteoglycan structure

1. core protein strand [peptide] has conserved S-G-X-G where GAG is attched

2. linkage most commonly thru trihexoside directly linked to O-Ser/Thr

3. many unique GAG chains can be dynamically attched to core protein

4. glycans are connected to membrane by protein or other anchors or just extracellular

“bottle brush appearance” of proteoglycans

linear structure of GAG chains allows negative charges of sugars to be furthest apart

[carbs]

GAG & its structure

GlycosAminoGlycan

• the polysaccharide attched

• linear chain of repeated disaccharides

• usually repeat unit is an amino sugar + acid sugar

• further modified post-attchment w/ epimers + sulfates

  • sugar acid makes neg charge → high water binding + unfolded structure

  • sulfate & alcohols provide “stickiness”

  • epimers change saccharide type

    • leads to lubricant or mucosal properties

in ECM, each proteoglycan is an…

aggregate

GAG: hyaluronic acid

• non-sulfated GAG w/ glucoronic acid + N-acetylglucosamine repeating disaccharide units

• largest GAG & retains water

• major component of synovial fluid [joint lubricant or shock absorber], vitreous humor of eye, & loose connective tissue

GAG: chondroitin sulfate

• glucoronic acid & sulfate-N-Ac-galactosamine

• most abundant GAG in body

• found in cartilage, bone, heart valves, tendons, ligaments

  • holds collagen fibers strongly

    • in joint supplments

• lost in osteoarthritis b/c of digestion of proteoglycans by metalloproteinases [in TGF-beta]

GAG: dermatan sulfate

• disaccharide of L-iduronic acid w/ N-Ac-galactosamine w/ sulfate group

• in skin, BVs, & heart valves

• Hunter’s & Hurler’s syndrome has lack of dermatan degradation in lysosome

• mitral valve prolaps has an increase in dermatan sulfate in heart valves

• I-cell disease charactered by absence of phospho-mannose being added

GAG: heparin sulfate

• disaccharide of iduronic acid + glucosamine

  • 2 sulfate groups attached

• Hunter’s & Hurler’s syndrome has lack of degradation in lysosome

• an anti-coagulant [enhances antithrombin III activity]

• Heparin is an intracellular component of mast cells that line arteries, esp in liver, lungs, skin

GAG: keratan sulfate [KS]

• disaccharide of galactose + N-Ac-glucosamine w/ sulfate

• KS-1 found in corneas & KS-II found in loose connective tissue proteoglycan aggregates w/ chondroitin sulfate

• also found in cartilage, bone, & IV disc

UDP-glycosyltransferases

synthesizes linker region of GAGS [anabolism]

GAG chain then elongated

GAG is degraded in…

lysosome after phagocytosis

*GAGs are unique so have special enzymes in lysozome to catabolize them

complex GAGs

• sulfo-iduronic acid

• sulfo-glucoronic acid

• glucosamine [N-Ac too]

• glucocuronic acid

iduronate 2-sulfatase

lysosomal enzyme that takes sulfate off of iduronic acid

alpha-L-Iduronidase

lysosomal enzyme that cleaves carb off & makes GAG 1 shorter

MPS Mucopolysaccharidoses

• several hereditary disorders caused by deficiency in GAG specific lysosomal hydrolases

• lack of heparan sulfate &/or dermatan sulfate degradation → lysosomal accumulation of GAGs in skin fibroblast or cells in blood

  • also semi-degraded GAGS detected in urine

• abnormalities in bones, joints, ligaments, & intellectual disability, height

Multiple Diseases

1. MPS 1 = Hurler’s

2. MPS 2 = Hunter’s

3. MPS 3 = Sanfilippo’s

4. MPS 4, 6, 7, based on other GAGs

loss of catabolic enzyme function [enzyme associated w/ each]

Hurler’s Syndrome [MPS I]

• alpha-L-Iduronidase deficiency

• corneal clouding, dvlpmnt disability, dwarfing, coarse facial features, upper airway obstruction, enlarged liver/spleen, progressive hearing loss

• degradation of dermatan sulfate & heparan sulfate affected found in urine

• deposition in coronary artery → ischemia & early death

• enzyme replacement & HSC transplant therapies

Hunter’s Syndrome [MPS II]

• iduronate sulfatase deficiency

• X-linked deficiency [ONLY MALES]

• NO corneal clouding

• mild-severe physical deformity [short neck, broad nose w/ flat bridge], hepatosplenomegaly, dvlpmnt disability, below avg height, aggressive behavior

• degradation of dermatan sulfate & heparan sulfate in urine

• enzyme treatment to slow progression

“X marks Spot for hunter”

S = sulfatase

Sanfilippo Syndrome [MPS III]

4 subtypes w/ diff GAG degradationn enzyme disrupted

1. Type A: Heparan sulfatase

2. Type B: N-Acetylglucosaminidase

3. Type C: Glucosamine N-acetyltransferase

4. Type D: N-acetylglucosamine sulfatase deficiency

• wide range of severity

• symptoms often appear after age 1 w/ slowing learning abilitis ~2-6

• coarse facial features

• sleep difficulty, behavioral issues, diarrhea, walking problems, stiff joints, below avg height

• enzyme test determines subtype

Dolichol phosphate

membrane anchor and transfers sugar molecules during assembly of glycoproteins in N-link pathway

interacts w/ UDP-GlcNac to elongate GAG

which are more diverse: N-glycans or O-glycans?

O-glycans

improper glycan transport [Golgi barcodes]

• lysosomal hydrolase enzyme being sorted for transport from golgi → lysosome

• substrate: N-acetylglucosamine uridine diphosphate

  • defect → I-cell disease

• key enzyme: N-acetylglucosamine phosphotransferase

mannose phosphate

denotes protein destined for lysosome

needed to form vesicle

I-cell disease

• inherited disorder of protein targeting

• defective enzyme: N-acetylglucosamine uridine diphosphate

• lysosomal hydrolases end up in blood instead of lysosome

  • diagnostic for I-cell [NO GAG]

  • no problems arise from extra enzymes in blood as lysosome as low pH

• b/c lysosomes lack proper enzyme, they fill up w/ substrates, esp. proteoglycans & glycoproteins

  • increased need for lysosome autophagy & decrease other organelles in autophagy

    • → poorly functioning mitochondria that increase reactive oxygen → cell death

• no treatment, death in early childhood

• problems catabolizing heparin, dermatan, & keratan sulfate

I-cell disease symptoms

• dvlpmnt disability & psychomotor impairments

  • loss of CNS cells

• skeletal issues & joint movement loss

  • loss of bone & muscle cells

• hepatosplenomegaly

  • from processing molecules for excretion & immune system degradation

I-cell imaging

• v similar to Hurler’s syndrome but more mild

• “I” = inclusion bodies

key differences

1. increased lysosomal hydrolase in blood

2. inclusion bodies in Fbs & B cells [image]

3. low activity of UDP-N-acetylglucosamine-1-phosphotransferase

Glycan in IgG

• maintains Fc domain in an open conformation allowing immune cell surface receptors to bind

• stabilizes IgG in blood

• in Fc but alters Fab binding strength

• imp. in making antibody-based therapies

Glycan Roles

• MHC-1 are glycoproteins w/ conserved N-linked glycosylation sites near peptide-binding groove

  • glycosylation essential for

    • proper folding & quality control

    • stability of MHC-I heavy chains

    • peptide loading & antigen presentation

• TCR is a glycoprotein

  • proper glycosylation ensures

    • stable TCR-MHC binding

    • signal transduction fidelity

    • efficienct immunological synapse formation

immunological synapse

CD8-TCR-MHC-I cluster btwn T cell and DC

*imp for creating a cytotoxic T cell

signaling & expansion of cytotoxic T cell formation

• CD28 on T cell bind DC to activate its protein

• inside CD8+ cell, Lck kinase activated

  • it phosphorylates ITAMs on CD3 [part of TCR complex]

    • this recruits ZAP-70 kinase, which initiates downstream signaling

• differentiation to cytotoxic T cell & clonal expansion occur, mediated mostly by IL-2

Fas is also known as

CD95

Bare Lymphocyte Syndome Type I

• v. rare condition w/ autosomal recessive inheritance

• diagnosed in childhood

• mutations occur in TAP1, TAP2, or TAPBP [tapasin] genes

  • unloaded MHC-1 complexes degraded and don’t reach cell membrane

    • incomplete T cell dvlpment

• recurrent internal bacterial infections

• labs show low CD8+ T cells, normal CD4+ helper T cells, & absent MHC-I

ZAP-70 deficiency

• defect in ZAP70 gene is a rare SCID like disorder w/ impaired adaptive immunity

• autosomal recessive & often diagnosed in 7th yr of life from recurrent infections

• mutations impair TCR signaling in CD8+ T cells, so they fail to develop

• failure to thrive, large lymph nodes, possible hepatosplenomegaly

• CD8+ cell lows, CD4+ cells normal but impaired

  • further testing will show poor T cell function upon stim.

CD28 overexpression in rheumatoid arthritis [RA]

• RA is a chronic autoimmune disease affecting about 1% of global pop.

• dysregulated overexpression of CD28 enhances T cell activation, promotes survival of autoreactive T cells, & amplifies inflamm cytokine prod, including IL-2

  • → joint inflamm & destruction

• Lab findings

  • positive rhematoid factor

  • increased ESR [erythrocyte sedimentation rate] or CRP

apoptosis

programmed cell death

• phosphatidylserine on inner membrane displayed on apoptotic bodies → macrophages & phagocytic cells to recognize it and engulf them

• 2 general types

  • intrinsic

    • due to internal damage or stress

  • extrinsic

    • due to membrane death receptors being acitvated

General events of apoptosis

1. initiation

   1. death signal received w/ pro-apoptotic signaling

   2. cytochrome c released

2. execution

   1. cell shrinks

   2. chromatin condenses [pyknosis]

   3. nucleus fragments [karyorrhexis]

   4. caspase cascade active

3. phagocytosis

   1. cell membrane blebs & apoptotic bodies

   2. done by macrophages w/o inflamm

Intrinsic Apoptotic pathway

ex. radiation induced DNA damage

1. due to DNA damage, p53 activated

   1. shifts balance toward apoptosis by increasing BAX & decreasing Bcl-2

2. mitochondrial outer membrane becomes permeable to BAX

   1. cytochrome c binds to Apaf-1 apoptosome & cuts procaspase-9 into caspase-9

3. caspase-9 cuts execution caspase-3 & others

4. macrophage engulfs apoptotic bodies

Fas-mediated Extrinsic apoptotic pathway

when CD8+ T cell recognizes a virus-infected cell via TCR it initiates apoptosis via Fas & FasL [& perforin/granzyme]

• when bound to infected cell, T cell expresses surface FasL [Fas ligand], which binds to FAS death receptor on infected cell

• → adaptor protein FADD [Fas-associated death domain] & procaspase-8

  • called death-inducing signaling complex [DISC] → active caspase 8

• active caspase 8 connects extrinsic pathway to intrinsic pathway since it cleaves Bid, a Bcl-2 protein

• truncated Bid [tBid] translocates to mitochondria, where it activates Bax, opening pore & promoting mitochondrial outer membrane permeabilization

BAX

pro-apoptotic protein

Bcl-2

anti-apoptotic protein

ALPS

Autoimmune Lymphoproliferative Syndrome

• rare genetic disorder characterized by defective lymphocyte apoptosis

• manifests in early childhood, diagnosis ~3 yrs

• caused by mutations in FAS gene

  • impairs abilitu of lymphocytes to undergo apoptosis → elevated lymphocyte count

    • large lymph nodes, liver, spleen

    • autoimmune attack of blood cellls

    • increased risk of lymphoma