Lack of K140 immunoreactivity in junctional epidermolysis bulls skin and keratinocytes associates with misfolded laminin epidermal growth factor-like motif 2 of the B3 short arm

1. VALENTINA ORTIZ
ZAPATA
FACULTY OF MEDICINE
UNIIVERSIDAD PONTIFICIA
BOLIVARIANA
2018

2. INTRODUCTION
◦ Highly specialized epithelial cells
◦ 85% of epidermis cells
◦ Originate in the stratum basale of
the epidermis where they are stem
cells
◦ Some migrate to the upper layers to
performe diferent functions
◦ FUNCTIONS:
◦ keratin production
◦ Protection of the oragnism from its
external enviroment
◦ Crossedshape glycoprotein with 3
polypeptides chains
◦ Genes: LAMA3, LAMB3 and LAMC2
◦ Major component of epithelial
basement membranes
◦ Assambled in the keratinocyte RE,
and then secreted to the
extracellular matrix
◦ Crucial for epithelial adhesion and
cell- extracellular matrix interaction
KERATINOCYTES
LAMININ

3. INTRODUCTIONK140
Anti-!3 chain
monoclonal antibody
mAbs in
immunofluorescence
analysis to evaluate protein
expression and distinguish
JEB subtypes
immunoreactivity
• Reaction antigen-antibody
• Ab’s function is to neutralize
bacterial toxins, binding to them
through the antigen
• Ag-Ab bind through non
covalent bonds forming a
complex.
• Immunochemistry techniques use
the Ag-Ac reaction to measure
elements.

4. OBJECTIVE
Through molecular characterization of
a patient with JEB, identify laminin
epidermal growth factor-like motif 2
of laminin !3 chain as the subregion
recognized by mAb K140

5. WESTERN BLOT
METHODS&
MATERIALS
principle
Analytic technique use
to determine proteins
in a specific sample
Molecular weight and
Ab binding specificity
uses
Evaluate K140’s
reactivity to patient’s
LM332 in keratinocyte
lysates sample
To determine if pAb
recognizes !3 chain in
the patient’s LM332

6. IMMUNOFLUORESCENCE
PRINCIPLE
USED TO:
Identify LM332 protein
expression in the patient´s
skin compared to a control
• Technique used to
identify the presence
of an specific
molecule (Ag)
• Ab labeled with a
fluorocrome
molecule
• Uses the Ag-Ab
reaction
• It could de direct
or indirect
METHODS&
MATERIALS

7. SEQUENCING
Principle:
Used to:
• Identify
mutations on
the patient’s
and his
parent’s
LAMB3 gene
• Determines the
order of the
nucleotides taht
make up the DNA
molecule
• They obtein DNA fractions , tag
them and finally separate them
by electrophoresis
METHODS&
MATERIALS

8. RT-PCR
PRINCIPLE
Can determine the
expression of genes in
various tissues
RNA is transcribed
into cDNA by the
reverse transcriptase
USED TO:
Study the patient’s
RNA to analyze the
mutation
consequences.
METHODS&
MATERIALS

9. RESULTS
◦ Patient’s skin shows
negative results using K140
compared to the control, in
addition staining with GB3
and BM165 was weaker on
the patient`s sample than
the control.

10. RESULTS
FIG 2
a) Patient’s keratinocytes show
negative staining for K140
compared to control.
◦ intense intracellular staining with GB3
b) Mutant LM332 and BiP show similar
staining pattern, supporting
retention in the ER
c) BiP expression is increased in the
patient’s cells

11. RESULTS
◦ Fig 3
a) Using K140,LM332 wasn’t
immunoprecipitated in the
patient’s media nor cell
lysates
◦ Increased levels of
unprocessed LM332 in
patients lysates (GB3) and
decreased levels of mature
LM332 in the media
b) Β3 chain was not
recognized by K140 but it
was detected by a pAb

12. RESULTS
◦ FIG 4:
a) The mother has a truncating
mutation on the intron 10, which is
also present in the patient`s DNA
b) There are shorter in-frame transcripts
with altered splicing of exon 10, the
shortest band correspond to
skipping of the entire exon 10

13. DISCUSSION
AUTOR CONCEPT YES OR NOT
Posteraro P, De Luca
N, Meneguzzi G et al.
In JEB-gen intermed forms caused bt LM332
mutations, missense variants and in-frame
transcripts predict synthesis of mutant laminin
chain subunits able to assemble in the
heterotrimer, which is then secreted and
deposited outside the cell, usually in reduced
amounts
Oslowski CM, Urano F. BiP is induced by accumulation of unfolded
polypeptides in the ER and implicated in their
retrograde transport across the ER membrane
for degradation by the proteasome, while
spliced XBP1 mRNA, which encodes an active
transcription factor, is a downstream effector
of the activated UPR response.
Murray LS, Lu Y,
Taggart A et al.
As chaperone drugs favour release of ER-
retained mutant proteins, we hypothesize they
may help keratinocytes to release ER-retained
LM332, leading to an increase in protein
secretion and disease amelioration.

14. CONCLUSIONS1.finding a way
to induce the
LM332 out of the
keratinocyte’s ER
might be a
treatment aim for
this kind of
patients
2. mAB K140
is crucial on
the diagnosis
of patients
with this
subtype of
JEB