Lecture 7 - Connective tissue disorders 1

Question 1

Basement membranes: abstract essential reading text

Answer 1

Basement membranes (BMs) are present in every tissue of the human body. All epithelium and endothelium is in direct association with BMs. BMs are a composite of several large glycoproteins and form an organized scaffold to provide structural support to the tissue and also offer functional input to modulate cellular function. While collagen I is the most abundant protein in the human body, type IV collagen is the most abundant protein in BMs. Matrigel is commonly used as surrogate for BMs in many experiments, but this is a tumor-derived BM-like material and does not contain all of the components that natural BMs possess. The structure of BMs and their functional role in tissues are unique and unlike any other class of proteins in the human body. Increasing evidence suggests that BMs are unique signal input devices that likely fine tune cellular function. Additionally, the resulting endothelial and epithelial heterogeneity in human body is a direct contribution of cell-matrix interaction facilitated by the diverse compositions of BMs.

Question 2

Layers of the gut

Answer 2

• Lumen
• Epithelium
• Connective tissue containing fibroblasts
• Smooth muscle containing circular fibres and longitudinal fibres
• Connective tissue containing fibroblasts
• Epithelium

Question 3

ECM: what is it and what does it do in the gut?

Answer 3

Extracellular membrane

Links with cells and bears the mechanical strain of tension and compression by skeletal filaments anchored to cell-matrix and cell-cell adhesion sites

Question 4

Collagen: how many types are there, which is the most abundant, and what do they do?

Answer 4

There are 28 different types of collagen

Most (more than 80%) is collagen I

Different collagens can form different types of ECM structures: stroma, tendons, basement membranes, etc

Question 5

Collagen 1: where is it found?

Answer 5

Found in tendons

Question 6

Collagen structure: what are the common features, what is the physical structure, and

Answer 6

All collagens have a repeating sequence rich in the amino acid proline, defined by the repeat Gly-X-Y: either Glycine-proline-Y or Glycine-X-hydroxyproline

Three collagen chains form a tight helical structure

Question 7

Hydroxylysine and hydroxyproline: what do they do and how are cross-links formed?

Answer 7

Form intrachain hydrogen bonds that stabilize the triple helix

Lysyl oxidase (LOX) deaminates hydroxylysine and lysine to generate reactive aldehyde groups, these form covalent bonds with other lysines and hydroxylysines to form intra/intermolecular crosslink

Question 9

Scurvy: what is it caused by and why does its cause have physiological issues?

Answer 9

Lack of vitamin C

Vitamin C is a cofactor for proline and lysyl hydroxylase, collagen produced has no cross-links - they fall apart

Question 10

Basement membranes: what are they, when do they appear, what do mutations in early basement membranes cause, and how diverse are BMs in adult tissue?

Answer 10

Basement membranes are the first recognisable ECM to form during metazoan development

They appear at the time when two distinct tissue layers form

Mutations in genes for these early BM result in no development beyond this stage

In adults, there are many variations of the BM in diverse tissues

Question 11

BMs: what do they do and what do their diseases manifest as?

Answer 11

Strong, often providing mechanical strength to tissues

Often manifest through loss of mechanical strength

Question 12

Collagen IV: what is it, how does it interact with collagen I, and what subunits are there to it?

Answer 12

Collagen specific to basement membranes

Several differences between it and Col I lead to its assembly into a 2-dimensional mat rather than fibres

N-term, coiled-coil, and C-term

Question 13

N-term of collagen IV: where is it found, how many amino acids is it comprised of, and what does it do?

Answer 13

N-terminal of collagen IV

~75, Cystein/lysine-rich

• Crosslinking of 4 triple helices via disulfide and lysine/hydroxylysine crosslinks

Question 14

Coiled-coil of collagen IV: where is it found, how many amino acids is it comprised of, and what does it do?

Answer 14

The main, central subunit of collagen IV

~1,200

Contains interruptions within the normal collagen Gly-X-Y repeats:
* (Gly-X-Y) (Gly-X-Y) (Gly-X-Y)n
* (Gly-X-Y) (Gly-A) (Gly-X-Y) G1G
* (Gly-X-Y) (Gly-A-A-A-A) (Gly-X-Y) G4G
These interruptions allow for Increased flexibility and sites for crosslinking

Question 15

C-term of collagen IV: where is it found, how many amino acids is it comprised of, and what does it do?

Answer 15

C-terminal of collagen IV

~230

• Drive triple helix formation
• Hexamer formation via end-end interactions

Question 16

NC1: what is it and what does it allow for?

Answer 16

A trimer formed from NC1 monomers

Hexamer formation at the protein C-term whilst a tetrameric trimer is formed at the 7S domain of the collagen - this contains extensive crosslinks holding the tetramer together

Question 17

How many collagen forms are there the potential for and how many are actually expressed?

Answer 17

56 possible combinations because of 6 collagen genes (a1(IV)-a6(IV))

But they only combine in three heterotrimers:
a1 a1 a2
a3 a4 a5
a5 a5 a6

Question 18

Glomerular BM: what is it, how thick is it, what cell layers does it connect, what properties does it have to aid its function, and what can go wrong if the wrong collagen is present?

Answer 18

Specialised BM that acts as a filter in the kidney

Forms a particularly thick BM compared with other tissues

Exists between two different layers of cells - endothelial cells of the blood vessels and podocytes (a specialised type of epithelial cell)

Col IV a3a4a5 is present: more inter/intrachain crosslinks than a1a1a2 - protecting it from both increased pressure and proteolysis from proteases in serum (allowing it to filter 90L/day)

Gradual proteolytic degradation of the GBM leads to disease if the wrong collagen is in the GBM

Question 19

Differences in renal basement membranes

Answer 19

In Bowman’s capsule, the a1a2a3 chain is present whereas in the GBM, a3a4a5 is present

Important to note, though, that a1a2a1 is expressed in development, a1a2a1 is expressed, but as the kidney matures, there is a switch in expression to the alternative a3a4a5 in the GBM

Question 20

Alports syndrome: what is it, what genes are mutated, what happens in those who suffer from it, where is it most common, and how severe is it?

Answer 20

X-linked mutation that is most common in men that is noted as a hereditary form of nephritis

COL4A5 gene most often but also both COL4A3 and COL4A4 are associated with the autosomal recessive form of Alports, these mutations are most often in the coiled coil domain

Most often X-linked, COL4A5 (85% of mutations in European families) is on the X chromosome, females are much less likely to suffer from this

The GBM breaks down, leading to kidney faiure

Severe in hemizygous males, although variable depending on the nature of mutations, end-stage renal disease (ESRD) by 20 years in early onset, and by 40 years in slower progressing, adult type. Can be very mild to non-existent in heterozygous females

Question 21

Collagen IV mutations: idek what to say just revise and learn lmao

Answer 21

Collagen IV genes are in pairs - arisen through gene duplications, these share a common, bi-directional promoter

Question 22

What types of disease onsets are present in Allports?

Answer 22

Early onset disease mutations include:
* Large gene rearrangements (~15%)
* Nonsense mutations and splice sites leading to truncations

Late-onset adult-type mutations include:
* Missense mutations disrupting Gly-X-Y repeats (~40%)
* Conserved cysteine residues affecting crosslinking

Question 23

What types of ways can disease function be monitored?

Answer 23

Transgenic - expressing an inserted gene - randomly

Knock-out - deleting a gene

Knock-in - Inserting a gene (endogenous) in a specific location

Question 24

COL4A3 knockout effect

Answer 24

COL4A3 knockout in mice develop progressive glomerulopathy - this phenocopies human autosomal dominant Alports

Question 25

ACE inhibitors: what do they do and what are their potential roles in helping alports syndrome?

Answer 25

The renin-angiotensin-aldosterone system controls blood pressure

Angiotensin converting enzyme (ACE) converts angiotensin I into angiotensin II

Angiotensin II induces vasoconstriction and increased blood pressure

ACE inhibitors such as Ramipril are used to treat hypertension and congestive heart failure

Reducing pressure in the glomerulus by vasodilation of efferent vessels increases the life span in Alports mice

Question 26

Can Alports be treated?

Answer 26

Genetically engineered mouse where Col4A3 can be turned back on again in glomerulus that has already developed

• Results show repair and return of function to defective GBM when new Col IV a3a4a5