What is MAN1B1 and how does it work?
MAN1B1 is the name of a gene that encodes an enzyme called alpha(1,2)-mannosidase. MAN1B1 protein resides in the Golgi where its function is to snip off the terminal mannose residue from the middle branch of a Man9GlcNAc2 glycan linked a maturing protein (Jakob et al., 1998). Removal of the terminal mannose is a quality control signal to the cell that a maturing protein is defective and must be returned to the endoplasmic reticulum. And from there escorted to proteasomes for recycling. This evolutionarily conserved cellular process is called endoplasmic reticulum-associated degradation, or ERAD.
MAN1B1 is a close relative of two other Golgi-resident alpha(1,2)-mannosidases, MAN1A1 and MAN1C1 (Rafiq et al., 2011). MAN1B1-CDG is a Type 2 congenital disorder of glycosylation. (See the SLC35A1-CDG and SLC35A2 Disease pages for more on Type 2 CDGs). Type 2 CDGs involve the later stages of N-linked glycosylation when maturing proteins have left the endoplasmic reticulum for the Golgi, where they grow a full glycan coat (Balasubramanian et al., 2019).
Like a robotic assembly line, a procession of Golgi resident enzymes adds to and subtracts from maturing proteins sugar building blocks, lengthening and pruning polysaccharide plumage as proteins pass from one Golgi lobe to the next. The MAN1B1 protein has several critical roles in cellular protein quality control, including functions in ERAD that are independent of mannosidase enzymatic activity.
So is MAN1B1 just an enzyme or does it wear multiple hats in cellular protein quality control? Turns out MAN1B1 is a “multifunctional gatekeeper” that has both enzymatic function and non-enzymatic function, in this case physically escorting proteins marked for ERAD to the exit door (Iannotti et al., 2014).
The MAN1B1 gene is conserved throughout the animal kingdom: in mice, zebrafish, flies, worms, budding yeast and fission yeast.
Clinical descriptions of MAN1B1-CDG
MAN1B1-CDG presents with three consistent diagnostic criteria: mild intellectual disability, facial dysmorphism and truncal obesity. In the last decade, whole exome studies of either patients with undiagnosed intellectual disability, or patients with genetically undefined congenital disorders of glycosylation, led to first confirmed diagnoses of MAN1B1-CDG.
Genetic and biochemical functional studies of four consanguineous families revealed that two pathogenic variants affect highly conserved amino acid residues (Rafiq et al., 2011). A second MAN1B1-CDG diagnostic cohort was described by longtime CDG researchers Drs. Jaeken and Matthijs (Rymen et al., 2013). They confirmed that MAN1B1 localizes to Golgi in the cell staining experiments. These experiments also revealed that Golgi compartments are disorganized in MAN1B1-CDG patient fibroblasts.
Loss of mannosidase activity in living cells can be confirmed in metabolic labeling studies of MAN1B1-CDG patient fibroblasts by comparing the levels of glycans with nine mannose residues versus eight mannose residues.
MAN1B1’s broader relevance
Industrial manufacturing of medical-grade recombinant proteins (biologics, enzyme replacements, and the like) in human cells still has technical hurdles to clear. The biggest challenge is figuring how to produce synthetic proteins with uniform glycosylation. This eliminates batch to batch variation and allows for scaleup of production. Recently a group discovered that by knocking out the three related mannosidases MAN1A1, MAN1A2, and MAN1B1, they could increase the simplify the glycosylation of two model enzyme replacement proteins (Jin et al., 2018).
Steps toward the clinic
Which animal models to make? Yeast are easy and cheap enough to try but their MAN1B1, called MNS1, localizes to the endoplasmic reticulum and mns1 mutants don’t have a robust growth defect (Camirand et al., 1991). A single 2012 paper about a potential worm model of MAN1B1 has not been extended (Wilson, 2012). Fly and zebrafish disease models are ripe for the taking. And of course a mouse model needs to be created.
Next steps for the MAN1B1 community are development of patient cell models, starting with fibroblast and moving onto to iPSC derived cell types, and in parallel development of functional and high-throughput assays and chemical probes to interrogate MAN1B1 enzymatic and non-enzymatic functions. The ability of other mannosidases to be activated to compensate for loss of MAN1B1 should be quickly put to the test as a therapeutic hypothesis. In parallel and as a hedge on assays and model systems that only read out MAN1B1 enzymatic activity, there should be a collaborative research efforts to conduct unbiased cell-based screens for drugs and novel compounds that restore normal Golgi structure or potentiate ERAD in MAN1B1-CDG patient fibroblasts. Pursuing targeted and unbiased approaches in tandem will lead to rational combination therapies. The potential for enzyme replacement therapy should be evaluated as well.
Finally, a few words about biomarker discovery. Any clinical trial protocol, starting with single patient compassionate use case studies, must include biomarkers to assess not only target engagement but also disease modification – even if that’s via a surrogate endpoint. A group has shown there was aberrant glycosylation of serum antibodies in ten MAN1B1-CDG patient blood samples (Saldova et al., 2015). Like serum transferrin, the glycosylation status of these serum antibodies could be monitored as a biomarker.
Balasubramanian M, Johnson DS; DDD Study. (2019). MAN1B-CDG: Novel variants with a distinct phenotype and review of literature. European Journal of Medical Genetics. 62: 109-114.
Camirand A, Heysen A, Grondin B, Herscovics A. Glycoprotein biosynthesis in Saccharomyces cerevisiae. Isolation and characterization of the gene encoding a specific processing alpha-mannosidase. (1991). Journal of Biological Chemistry. 266: 15120-7.
Iannotti MJ, Figard L, Sokac AM, Sifers RN. (2014). A Golgi-localized mannosidase (MAN1B1) plays a non-enzymatic gatekeeper role in protein biosynthetic quality control. Journal of Biological Chemistry. 289: 11844-58.
Jakob CA, Burda P, Roth J, Aebi M. (1998). Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. Journal of Cell Biology. 142: 1223–1233.
Jin ZC, Kitajima T, Dong W, Huang YF, Ren WW, Guan F, Chiba Y, Gao XD, Fujita M. (2018). Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose-type N-glycans. Journal of Biological Chemistry. 293: 5572-5584.
Rafiq MA, Kuss AW, Puettmann L, Noor A, Ramiah A, Ali G, Hu H, Kerio NA, Xiang Y, Garshasbi M, Khan MA, Ishak GE, Weksberg R, Ullmann R, Tzschach A, Kahrizi K, Mahmood K, Naeem F, Ayub M, Moremen KW, Vincent JB, Ropers HH, Ansar M, Najmabadi H. (2011). Mutations in the alpha 1,2-mannosidase gene, MAN1B1, cause autosomal-recessive intellectual disability. American Journal of Human Genetics. 89: 176-82.
Rymen D, Peanne R, Millón MB, Race V, Sturiale L, Garozzo D, Mills P, Clayton P, Asteggiano CG, Quelhas D, Cansu A, Martins E, Nassogne MC, Gonçalves-Rocha M, Topaloglu H, Jaeken J, Foulquier F, Matthijs G. (2013). MAN1B1 deficiency: an unexpected CDG-II. PLoS Genetics. 9: e1003989.
Saldova R, Stöckmann H, O’Flaherty R, Lefeber DJ, Jaeken J, Rudd PM. (2015). N-Glycosylation of Serum IgG and Total Glycoproteins in MAN1B1 Deficiency. Journal of Proteome Research. 14: 4402-4412.
Wilson IB. (2012). The class I α1,2-mannosidases of Caenorhabditis elegans. Glycoconjugates Journal. 29: 173-179.