Sebastian table tail, Product (kit), Patient selection, Catalytic pathway, GSL flux, GluCer / GalCer isoforms, Gangliosides

Sebastian 20201124 (continued)

Henriques et al	2017	Tissue lysate	b-NPG (commercial kit)	20 min incubation, measure	CBE treatment reduced activity		~100-220 U/μg protein
Prevail report	2020	CSF	?	?	Activity increased at 4 months after treatment	정상치라함	1.1 – 8.1 μmol/l/d (근거무)

Product (kit)

Company	Sigma-Aldrich	SEMA4	ANASPEC, SensoLyte® Red Glucocerebrosidase (GBA) Assay Kit This is for research, not for human!	LABCORP	ARUP
	https://www.sigmaaldrich.com/catalog/product/sigma/mak129?lang=ja®ion=JP	https://sema4.com/products/test-catalog/acid-%CE%B2-glucosidase-gaucher-disease/	https://www.anaspec.com/products/product.asp?id=59067	https://www.labcorp.com/tests/451780/gaucher-disease-enzyme-analysis-glucocerebrosidase	https://ltd.aruplab.com/Tests/Pub/2014459
	colorimetric tests β-glucosidase activity, β-Gurukoshi Daze is p - nitrophenyl -β-D- glucopyranoside hydrolyzed, available colorimetric (4 05 nm), anti-producing product that is proportional to the existing β-glucosidase activity is measured by the response. One unit of β-glucosidase is the amount of enzyme that catalyzes the hydrolysis of the substrate at pH 7.0 at 1.0 μmole per minute. Sebastian started from this	measured with a lysate from the patient's (WBC) by a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method. Specimen requirements: Anticoagulated blood: 5-10 mL in sodium heparin tube (green top) or 1-2 leukocyte pellets	Fluorogenic substrate. The GBA substrate provided in the kit releases the red fluorescent dye resorufin upon Glucocerebrosidase cleavage with absorption/emission maxima at 570 nm/610 nm.	fluorometric enzyme assay using 4-methylumbelliferyl-β-D-glucosida as substrate, whole blood is used	Quantitative Fluorometry, whole blood
Turnaround time		7 days		7-13 dyas
These are 'research only' so not clinically validated, or FDA-approved? Not companion-diagnositc (근데 위기술보면, whole blood에서, 진단치도 있고, normal치도 있는것 같은데?)

• Patient selection: As whether GBA activity is actually reduced is not established in some GBA variants, we may confirm GBA deficiency in participants if we measure GBA activity in individuals and pre-define cut points.

• Additionally, Patient enrichment strategy The hypothesis that GBA-PD patients with significantly reduced GBA activity (<x %) may be validated clinically. I envisioned a clinical scenario below for GBA-targeting treatments. A question would be: would we need any preclinical support to work on the hypothesis in addition to our planned preclinical POC plan? In GBA-PD trial, upon entry to the trial, categorizing the participants according to GBA activity level on predefined cut-points, and subsequent enrichment of subpopulation based on differentiated treatment response-if any.

Catalytic pathway

• Ceramide가 다시 lysosome 밖으로 나가서 → GCS에 의해 GlcCer (UGCG 일걸)가 됨.

• GlcCer는 ① lysosome안에서 GBA에 의해 Ceramide만들기도 하지만, ② (Acid ceramidase=ASAH1, 에 의해) GlcSph로 되기도 함. → 이 GlcSph 가의해) Sphingosine 되는 것임 → 이후 다른 sphingolipid 로 진행될걸 (glucosylceramide hydrolysis accounts for 50-90% of sphingolipid production.)

• GBA 막으면 → ↑ glucosylceramide & ↑↑ glucosylsphingosine (Glucosylceramide 로부터 변환되는 것들까지 합쳐지니 더 많이 증가하겠다)

• GCS 막으면 → ↓ glucosylceramide & ↑ (정도?) glucosylsphingosine (더 느리게 감소하겠다)

Controlling the Flow. GBA catalyzes a crucial step in the lysosomal breakdown of glucosylceramide-based glycosphingolipid/GSL synthetic pathway,

• Catalyzes at the cytosolic surface of the Golgi, the initial step of the glucosylceramide-based glycosphingolipid/GSL synthetic pathway,
• the transfer of glucose from UDP-glucose to ceramide → to produce glucosylceramide

GluCer (Glucosylceramide) isoforms

• GluCer is comprised of different isoforms owing to differen fatty acid moieties.
• GluCer isoforms are isobaric structural isomers of galactosylceramide (GalCer) isoforms
• GalCer has similar chemical properties as GluCer but is not a substrate of GCase.
• Both species can be differentiated only by the axial or equatorial configuration of one hydroxyl group

Normal range

• CSF : 4.5—5.9 5.9 μg/mL ng/mL의 typo?(venglustat P2 LEAP trial)

glucosylsphingosine (lyso-Gb1), a downstream metabolic product of glucosylceramide, a deacylated form of glucosylceramide, is also degraded by the glucocerebrosidase. β-cerebrosidase enzyme catalyzes the metabolism of glycosphingolipids

Figure caption (under the GluCer / GalCer chemical-structure pair):

Structures of (A) glucosylceramide (GluCer) and (B) galactosylceramide (GalCer). Both molecules are comprised of isoforms according to different fatty acid chains. The cleavage site of the glucocerebrosidase (GCase) enzyme is indicated on the molecules. Red circles indicate the hydroxyl group differentiating GluCer and GalCer by its axial or equatorial configurations. Glu = glucose, Gal = galactose.

Gangliosides

Source: (Kolter 2012, PMID 25969757)

Conclusion: gangliosides are formed from GlcCer and ceramide, so it is hard to predict whether GBA loss will lead to increase or reduce gangliosides.

Expression	most abundant GSLs in the CNS, predominantly in the nervous system where they constitute 6% of all phospholipids.[3], especially abundant in the brain, where their occurrence in the grey matter is about 5-fold higher than in white matter Gangliosides are also found in serum. There, especially GM3, GD3, GD1a, GM2, GT1b, sialylneolactotetraosylceramide (Figure 10), GD1b, and GQ1b are present, where about 98% of them are transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%) [69].
Location	a component of the cell plasma membrane that modulates cell signal transduction events, and appears to concentrate in lipid rafts
Definition & structure	complex GSLs (glycosphingolipid), composed of a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids (e.g. n-acetylneuraminic acid, NANA) linked on the sugar chain 즉 다음 세 부분의 결합체

Uncertain Spans

location	transcription	uncertainty
LABCORP method cell	4-methylumbelliferyl-β-D-glucosida	reads as written; the trailing se of glucosidase may be clipped or omitted in the source.