Carrier screening (continued), Structure / Function / Turnover / Expression / Lifecycle of GBA, activity (‘BGL test’)

Carrier screening (continued)

• https://sema4.com/products/test-catalog/expanded-carrier-screen-283/
  • 283 GENES
  • may be easily customized to address specific clinical needs.
  • Turnaround time: 14 days from receipt of specimen
• Gene sequencing can identify over 99 percent of Gaucher disease carriers.

Single gene testing

full sequencing of all exons of GBA1

screening for 5 to 10 specific variants

screening for 2 to 4 specific variants

Structure of GBA protein (EC 3.2.1.45)

• 497-amino-acid membrane-associated protein with a 39-amino-acid leader sequence and five glycosylation sites [4, 5].
  • My thesis: Mature GBA is a glycoprotein consisting of 536 amino acids (Rocha et al., 2015b), derived from a precursor that is proteolyzed and undergoes lysosomal trafficking following its synthesis on free ribosomes in the cytoplasm (Qin et al., 2013).
• a calculated molecular mass ranging between 55 and 69 kDa depending on the number of occupied glycosylation sites.
• The protein consists of three domains.

	Domain 1	Domain 2	Domain 3
Resides	residues 1-27 and 383-414	residues 30-75 and 431-497)	(residues 76-381 and 416-430
	an antiparallel β sheet flanked by an amino terminal strand and a loop. This domain also contains two disulfide bridges (residues 4-16 and 18-23) which are required for correct folding of the protein [56].	n immunoglobulin-like domain comprised of two closely associated β sheets	catalytic domain, and is a (β/α)8 triosephosphate isomerase (TIM) barrel with three free cysteines at residues 126, 248 and 342
Example of mutation		L444P, is located in the hydrophobic core of the Immunoglulin-like domain (domain II). (catalytic site is intact) N370S is located in the longest helix in the protein (helix 7) at the interface of domains II and III, but is too far from the active site to participate directly in catalysis. Any mutation in this domain may produce an unstable protein due to disruption of the hydrophobic core and altered folding of this domain [57].	active site in GBA protein Glu340 (E340).(MY thesis) (Romero et al. 2019, PMID)The active site, containing two catalytic residues—namely, E340 (catalytic nucleophile) and E235 (acid-base residue)—lies in a cavity formed at the center of the TIM barrel motif surrounded by residues R120, D127, F128, W179, N234, Y244, F246, Y313, C342, S345, W381, N396, F397, and V398. Cf) The catalytic residues of an enzyme 의 정의: as the amino acids directly involved in chemical catalysis. They mainly act as a general acid-base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state. Cf) Tim barrel: a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone.[1], TIM barrels are ubiquitous, with approximately 10% of all enzymes adopting this fold.

• transition electron microscopy studies have clearly shown that GCase has a butterfly-shaped dimer structure both in solution and as a crystal,

Function of GBA protein

• GCase generates ceramides from GlcCer molecules in the outer part of the skin, a process essential for optimal skin barrier property and survival.

Turnorver of GBA protein

• The turnover of GBA is not cell-specific and it has been calculated from the experiments in cultured human fibroblasts from normal subjects that GBA is synthesized at a rate of 1.8 ng GBA/h/mg cell protein and that 1.4% of the enzyme molecules present are destroyed in that time (Das et al., 1986).

Expression of GBA

• Transcriptome data from human astrocytes and human neurons show that more GCase is expressed in astrocytes than neurons (Zhang et al., 2016).
• GCase is ubiquitously expressed, throughout all inspected brain cortical areas and subcortical structure
• But regional variations have been consistently found,

		Expression level
Human	IHP (Dopeso-Reyes et al. 2018, PMID) Histology	High	cholinergic neurons from the nucleus basalis of Meynert, dopaminergic cells in the SNpc, serotoninergic neurons from the raphe nuclei, as well as in noradrenergic neurons located in the locus ceruleus
		moderate	in a number of areas within the paleocortex and archicor tex,such as the entorhinal cortex and the hippocampal formation, respectively

Lifecycle of GBA

cytosol	ER		ER		Golgi		Lysosome
translation from mRNA	Protein folding & QC	→	GBA is recognized by trafficking receptor LIMP2 → packaged in a vesicle	The vesicles are trafficked to golgi	Further processing	Trafficked by the GCase transporter lysosomal integral membrane protein-2 (LIMP2), encoded by the gene SCARB2 [6]	the vesicle empties its cargo→ GBA forms a complex with co-activator Saposin C → hydrolyzes its substrate glucosylceramide on the surface of the lysosomal membrane.
	undergoes N-linked glycosylation on four asparagines, after which it is subject to ER quality control (ERQC).

activity (‘BGL test’)

Hypothesis: Intracellular trafficking of GBA, MOA of silent chaperone ^{(Takeda confidential)}

1. In ER, GBA forms a dimer and binds to LIMP-2 dimer. Cell 131, 770. and PNAS 113, 3791
2. After trafficking into lysosome, LIMP-2 dissociates the GBA dimer under the acidity. Cell 131, 770. and PNAS 113, 3791
3. GBA forms a monomer by the interaction of Sap C existing only in lysosome. Biochemical and Biophysical Research Communications 457, 561.
4. Monomeric GBA shows enzymatic activity. (The activity is enhanced by Sap C.)

• 18 Medscape) glucocerebrosidase activity in peripheral blood leukocytes. a standard blood test called a β-glucosidase leukocyte (BGL) test
• Most major labs, such as Counsyl, Labcorp and Quest Diagnostics, are familiar with a β-glucosidase leukocyte (BGL) enzyme assay and have historically been able to perform the test.
• A finding of less than 15% of mean normal activity or < 8.7 nmol/h/mg protein (https://gaucherdiseasenews.com/bgl-test/), or 4.6 - 12.0 nmol hydrolyzed/hr/mg protein (ARUP) is diagnostic of GD.
• Heterozygotes generally have half-normal enzyme activity, but as much as 20% overlap with activity levels of healthy controls has been reported (Essabar, 2015 #847),