4120 bcmb Test 2

role for lipids and carbohydrates esp at cell surface

what did the first central dogma ignore

expanded dogma

structure, biosyn, and biology of glycans and glycoconjugates

what is glycobiology

There has failed to be any living cell devoid of a dense and complex array of cell surface glycans

ubiquitous glycosylation

role fore carbohydrate in the societal interactions of cells in tissues (glycocalyx)

Predominant cellular distribution of glycans implies

glycoproteins or glycoconjugates

nearly all biologics are

a hormone responsible for stimulating the maturation or red blood cells

core protein MW: 18 kD

erthropoietin

3 N-linked glycans and at least 1 O-lined glycan, MW of secreted protein = 34 kD

Erythropoietin produced in CHO cells also carries 3 N-linked glycans and 1 O-linked glycan (AMGEN)

erythropoietin purified from human tissues carries

Erythropoietin produced in CHO cells also carries 3 N-linked glycans and 1 O-linked glycan

blockbuster drug

claimed improved quality of life

showed to have lower quality of life in cancer patients

AMGEN Epogen

increased N-linked glycosylation (basically more sites for it)

native Epo = 3 N-linked sites

AMGEN = 5 N-linked site

what enhances efficacy of erthropoietin

single sugars

typically 4-10 sugars

monosaccharides, oligosaccharides, polysaccharides

isomers

Common monosaccharides found in vertebrates are mostly structural

Glucose (D-glc), GlcNAc, D-Galactose (Gal), GalNAc, D-Mannose (Man), D-Xylose (Xyl), D-Glucuronic acid (GlcA), L-Fucose (Fuc), NeuAc

common monosaccharides (8)

potential for high information content

what is inherent in glycan structure?

hexoses: circles

N-Acetylhexoasmines: squares

Hexosamines: squares divided diagonally

Acidic sugars: diamonds

nomenclature for glycans

Shape and electrostatics

what drives biology?

post-synthetic modifications

what are commonly built into polymers and increases structural diversity?

No

Are oligosaccharide structures encoded in DNA?

Glycosyltransferases

Glycosidases

Availability of monosaccharide and activated forms

Organelle distribution and function

Correct synthesis of carbohydrates depends on?

Glycosylation is very sensitive to subtle shifts in the environment

Consequence of there being no template for Carbohydrates?

Glycoproteins

Glycolipids

Proteoglycans

classes of glycoconjugates

protein + carbohydrate

most proteins of biological and therapeutic significance

eg. TPA, EPO, LH, Immunoglobulins

Glycoproteins

Lipid + Carbohydrate

important for tissue and cell type markers

eg. blood group antigens, tumor associated antigens

Glycolipids

complex of a protein and one or more polysaccharides

known as glycosaminoglycans (GAGs)

immense structural diversity, pleiotropic functions

proteoglycans

N

classes of animal glycans

Asn

Major class of N-linked glycans attach to N of which aa side chain

Dolichol

large precursor of Asn side chain glycans is built on

subsequent trimming and re-extension in ER and Golgi

what is done to generate high-mannose, hybrid, or complex glycans? And where does it occur?

hybrid/complex glycans

loss of what is embryonic lethal?

Ser/Thr

Major class of O-linked glycoproteins attach to O of which aa(s) side chain

GalNAc to Ser/Thr residues by one of 20 or so polypeptide GalNac Transferases

mucin type of o-linked glycoproteins initiated by attachment of what to what?

No, sequences enriched in S/T residues and frequently flanked by P

Other types of O-glycans include O-Man, O-Fuc, O-Glc

is consensus of O-linked strong?

Mucins

what are heavily O-glycosylated

contains tandem repeats (8-169 aa)

rich in proline, threonine, and serine (PTS domains)

Apmucin

80

glycosylation constitutes as much as __% of mass and tends to be clustered

epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts

mucins are expressed by

protective barriers

mucins are _____ for epithelial cells

Lubrication

modulate infection

• receptors for bacterial adhesins

• Secreted mucins can act as decoys

Barrier against freezing

• antifreeze glycoproteins

• [Ala-Ala-Thr] w/ core 1 disaccharides

what do mucins provide for epithelial cells?

epithelial cancers (lung, pancreatic, ovarian, colon etc) are characterized by altered O-GalNAc glycan sialylation

Oglycans made by these cancer cells suppress immune surveillance

Palleon is targeting altered sialylation (by removing sialic acid) as a way to enhance immune cell killing of tumor cells

clinical relevance of mucin type O-glycans

differ based on composition of core glycan

gangliosides are major components of lipid rafts and modulate receptor signaling

*Many glycolipids are receptors for pathogens and toxins

major types of glycoconjugates; glycosphingolipids

GM3 deficiency

missense mutation (E332K) in ST3GalV (GM3 Synthase) middle of S-motiff

profound intellectual disability

failure to thrive

seizure disorder

neural crest defects: midface hypoplasia, scattered dermal hyper- and hypopigmentation

salt and pepper syndrome

Gangliosides

______ are essential for normal brain function and post-natal development in humans

Allelic disorder: truncation (R232X) in ST3GalV, b/w L- and S- motiff

Old Amish Infantile Epilepsy Syndrome

GPI added sequences that share physicochemical features, but little aa identity

GPI anchors

alternative way to tether proteins at membrane (and release—how?)

what do GPI anchors provide

lipid rafts

GPI-anchored proteins are enrinched in

acylation, mannose, glucosamine, and inositol

structural variation primarily arises from

RBCs, complement-mediated lysis

GPI-linked proteins protect ___ from ___

Impaired quality of life, Thrombosis, Anemia, Smooth muscle dystonia

urine enriched in hemoglobin breakdown products (dark to light yellow/red over the day)

Paroxysmal Nocturnal Hemoglobinuria

cytokine and chemokine

initated by carbh. recog b/w leukocytes and enothelial cells

what recruits leukocytes to site of inflammmation

selectins PSGL1 VLA4: capture of leukocytes

leukocytes to site of inflammation starts with

selectin (L-selectin)

lymphocytes express their own

PNAds (peripheral node adressins): mucin-type proteins expressed on high endothelia cells

at HEV of lymph nodes

L-selectin bind to

O-linked glycans on PNAds

MECA-79 recognizes 6-sulfo sialyl Lewis X

what does antibody recognized for L-selectin on lymphocytes

O-linked glycans on PSGL-1 protein expressed on lymphocytes

P-selectin recoginzes

find targets, phagocytose, and kill via halogenation w/ hypochlorite (bleach) derived by oxidative burst

when leukocytes in cell what happens

ppp pathway provides NADP+

NADPH oxidase

Superoxide dismutase

Myeloperoxidase

what enzymes involved in leukocytes part in cells

superoxide dismutase, catalase, and other peroxidase

what enzyme provides protection against oxidative burst

complement activation

what’s another way to attack pathogens

attack mech using cirulating protein and protease precusors

Goal to get complement proteins to come together and form whats needed

complement-mediated lysis

come together to activate C3 in a protease cascade

C3 wants to be activated; cleaves itself

B, D, and P stabilize self cleaved form of C3

alternative pathway for complement lysis

Factor H: binds factor B

Factor I inhibits by cleaving C3b into inactive form

breaks on lysis

downstream complement proteins

C3 cleavage leads to

MBL: (mannose) induced by recognition of carbohydrate residues on susceptible pathogens

carbohydrate binding domains

binds yeast mannans and other polygmeric glycans

Ficolin protein: binds bacterial glycans terminated in LacNAc and other polymeric glycans

recognize patterns (kinda like TLRs)

lectin pathway

MASP (mannan Serine Protease)

mannose binding recruites

activate C3 and other complement components in similar manner to classic and alternative pathway

high-mannose glycans on C3 only recognized when cleaved to C3b

MASP proteases

secretes ficolin

tracheal epithelium

deuterostomes from shark to primate, but no protostomes have it

not for sure

why so long to evolve adaptive immunity

small box, variola virus

sir edward jenner (attacked virus by snorting it)

what virus killed 30% of infected and permantely scarred

milkmaids came in contact with cow pox but were resistant to smallpox

forced cowpox to humans via cut in skin

eventually smallpox eradicated by vaccination

what did edward jenner note

lymphocytes cullular and humoral immuner response confer long lasting protection

production of binding recognition proteins that recog limitless molc structure (antigens)

generation of naive lymphocyte precursors

mech for selecting and amplyfing recmbined clones (learning)

memory

mech for tissue surveillance

components of adaptive immunity

(b cell → plasma & memory)

(t cell → Th cell & Tc cell)

NK cell

lymphocytes →

extensive filopodia

lymphocytes generally possess very little cytoplasm but project

born in bone marrow but migrate to thymus

undergo selection before maturing in circulation, lymphatic tissues, and at site inflammation

T-lymphocytes

periphery CD8 (cytotoxic t cell)

CD4+ (helper cells)

thymus Tp →

participates in macrophage activation, promotes cellular activity, produces IFNy

T helper 1 (Th1)

promotes humoral immunity

Produces IL-4, IL-5, and IL-3

Th2 cell

born in marrow, differentiate in lymphatic tissues

become antibody producing plasma cells

B cells

25

total B-cell account for __% of circulating lymphocytes

signaling to support commitment of resources

transition from B-cell to plasma cell requires extensive

cells of innate pathway via antigen presentation and cytokine support (Dendritic and Macrophages)

T cells activated by

dendritic cell population of the dermis; professional antigen presenting cells

Langerhans cells

in liver, population of tissue resident macrophages, antigen presenting cells

Kupffer cells

b cells, t cells, dendritic and macrophages, all somatic cells

cell specific receptors relavent to adaptive immunity

immunoglobulin (somatic rearrangement)

BCR (somatic rearrangement)

cytokine and chemokine receptors (germ-line encoded)

b cell receptors

TCR

Cytokine and chemokine receptors

selectins (germ-line encoded)

t cells

TLR (germ line-encoded),

MHC (germ-line encoded),

Cytokine and chemokine receptors

dendritic cells and macrophages

MHC (germ-line encoded)

somatic cells receptors

heavy and light chains

disulfide bonds (inter-chain & intrachain)

Immunoglobulin structure

Ag binding

Binding to Fc Receptors

Complement binding site

immunoglobulin fragments (structure/function relationships)

essential for antibody function

IgG1 glycans are

MBP, leading to complement activation

under-galactosylated IgG1 is recognized by

Papain

Fab: -ag binding, valency, specificty determined by V_H and V_L

Fc: effector functions

immunoglobulin fragments (structure/function relationships)

IgG - Gamma (y) heavy chains

IgA - Alpha (a) heavy chains

IgM - Mu (u) heavy chains

IgD - Delta (δ) heavy chains

IgE - Epsilon (ε) heavy chains

human immunoglobulin classes

major serum Ig (systemic immunity)

major Ig in extravascular spaces

placental transfer (passive immunity for newborn)

fixes complement

binds to Fc receptors

monomer

IgG

serum monomer; secretions (sIgA) (dimer, J chian, secretory component)

2nd highest serum Ig

major secretory Ig (mucosal or local immunity)

does NOT fix complement (unless aggregated)

binds to Fc receptors on some cells

IgA

pentamer, extra domain, J chain

3rd highest serum Ig

first Ig made by fetus and B cells

B-cell receptor as monomer

fixes complement

IgM