About CD99 D4와 patholog 관계 Cell surface glycoprotein leukocyte antigen. 세포 내 물질 이동에 관여. TCR-CD3 complex와 더불어 T-cell을 activation 시키는 co-stimulatory molecule로 작용하여 thymocyte와 T-cell을 activation 시킨다.

: paired Ig-like type2 receptor PILR : paired Ig-like type2 receptor D4의 ligand로 알려져 있음. PILR이 흉선세포의 세포사멸, 특히 death by neglect에 관여한다는 것이 증명. 대부분의 mammalians 에 존재를 하며, inhibitory and activating isoform 둘 다를 가진 형태. inhibitory: PILR α ->possesses an ITIM( immunoreceptor thyrosine-based inhibition motif) in its cytoplasm domain activating :PILR β ->dose nor have an ITIM but transduces activating signals by associating with ITAM ( immunoreceptor thyrosine-based –activation motif ) bearing DAP-12 adapter molecules.

Result 1. post transcriptional modification of CD99 affects the recognition by PILR

->human, mouse CD99 : possess O-glycosylated type I transmembrane protein do not possess N-glycosylation site ->O-glycosylation of CD99 might affect the recognition by PILR

Result 2. involvement of O-glycosylation on CD99 in the recognition by PILR GalNAC-α-O-benzyl : sugar analog that specifically inhibits O-glycosylation

-> CD99에서 glycosylation은 PILR 에 의한 CD99의 recognition에 중요한 역할을 한다. Western blot

Result.3 identification of O-glycosylation sites on CD99 involved in the recognition by PILR O-glycosylation is generated on serine or threonine residues. (ref.20)

Using the NetOGlyc3.1 algorithm Four serine or threonine residues were predicted to be possible O-glycosylation sites

B. Transfected into Ba/F3 cell stained with PILR-Ig & anti-CD99 mAb c B. Transfected into Ba/F3 cell stained with PILR-Ig & anti-CD99 mAb c. western-blot ->lysate of 293T cells transfected with CD99W and CD99M were separeted by SDS-PAGE and were blotted with anti-CD99 mAb.

Result.4 role of sialic acids on the recognition of CD99 by PILR. PILR에 의한 amino acid의 4~10%가 glycan의 sialic acid를 recognition하는 Sialic-acid-binding Ig-like lectin family와 동일하다는 이전의 분석. -> PILR에 의한 CD99의 recognition에 sialic acid가 영향을 미칠까?

Sialic acid : 악하선(顎下腺)의 뮤신에 많이 함유. 화학구조는 피루브산과 만노사민의 알돌축합체인 노이람산이 n-아세틸 또는 n-글리콜, 때로는 o-아세틸화 한 것이다. 인플루엔자바이러스에 의한 혈구응집 작용은 당단백질인 시알산에 의하여 저해된다는 것이 밝혀졌다. 시알산은 이들 당단백질의 비환원성 말단에 존재한다. 시알산은 케토기 ·글리코시드결합에 관여하고 있으므로, 카르복시기가 유리상태에 있다. 생체 내의 pH에서는 이 카르복시기는 거의 해리하여 음전하를 가지고 있어서 혈구응집 반응 등 중요한 생리적 의의가 있다. * neuraminidase ( 뉴라민산가수분해효소) : 뉴라민산을 가수분해하여 sialic acid를 분리하는 효소를 말한다.

A. Ba/F3 cells transfected with wild-type CD99 or CD99W-T45A-T50A were treated with neuraminidase->stained with PILR-Ig or anti-CD99 mAb

Result.5 modification of O-glycans on CD99 by core 2 branching enzyme inhibits the recognition by PILR Core1 and 2 structures are the main components of O-glycans. Core 2 O-glycan branch is generated by C2GnT, and there are several isozymes of C2GnT. O-glycans의 생합성 -> if C2GnT가 없다면 core1 structure인 Gal-1.3 GalNAc-R 은 2가지 서로 다른 sialic acid 전이효소에 의해 종결되어 4당류를 만들게 됨. If C2GnT가 충분하게 발현되면 core1 은 core2 structure인 GalNAc-1,6 (inhibit o-glycosylation) 로 전환.

A. Ba/F3 cell에 mock or CD99 transfect ->core2 branching enzyme / control vector 를 transfect ->control Ig / PILR Ig / anti-CD99 mAb 로 stained M : transfect with control vector C2 : transfect with core2-branching enzyme

->CD99 is expressed on T cells upon activation. CD99 O-glycan 내 core2 branch 가 PILR에 의한 recognition을 inhibit한다. -> then,,,PILR에 의한 CD99 recognition은 C2GnT 에 의해 regulate되어지는 것인가?? C2GnT is expressed in B cells and is involved in the generation of the B220 epitope on CD45 (ref.29) ->CD99 is expressed on T cells upon activation. =>compared recognition of CD99 expressed on activated T cells and B cells by PILRα-Ig or PILRβ-Ig

PILRα 에 의한 B cells가 아닌 activate 된 CD4+ T cells의 recognition. Naïve CD8+ T cells express unknown ligands for PILRs. ->B cells 내 C2GnT 의 expression이 PILR에 의해 CD99의 recognition을 regulate한다.

Cytotoxicity : (세포 독소에의한 세포 독성) Result 6. involvement of O-glycan on CD99 in the cytotoxicity mediated by NK cells. Cytotoxicity : (세포 독소에의한 세포 독성) is the quality of being toxic to cells. Examples of toxic agents are a chemical substance or an immune cell. Cytotoxicity can be measured by the MTT assay, Trypan blue (TB) assay, Sulforhodamine B (SRB) assay, WST assay and clonogenic assay

NK cell : were purified from splenocytes of C57BL/6 mice NK cell-mediated cytotoxicity assay ( :performed by using PKH2 fluorescence dye-labeling method.)

(참고)

summary 1.CD99의 posttranscriptional modification은 PILR에 의한 recognition에 영향을 미친다. 2.CD99에서 O-glycosylation은 PILR에 의한 CD99의 recognition에 중요한 역할을 한다. 3.CD99의 O-glycosylation sites의 identification은 PILR에 의한 recognition에 관계한다. 4.sialic acid is involved in the recognition of CD99 by PILR. 5.core2 branching enzyme으로 인한 CD99 O-glycans의 modification은 PILR에 의한 recognition을 inhibit한다. 6.cytotoxicity 에서 CD99 O-glycan의 involvement는 NK cell에 의해 mediate 된다.