The ABCs of the atypical Fam20 secretory pathway kinases

The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes. The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes. The study of protein phosphorylation began as early as 1883 to 1900, when phosphorous was detected in milk casein (1Hammarsten O. Zur Frage ob das Caseïn ein einheitlicher Stoff sei.Z. Physiol. Chem. 1883; 7: 227-273Google Scholar) and egg-yolk phosvitin (2Leven P.A. Alsberg C. Zur chemie der paranucleinsaure.Hopper-Seyler’s Z. Physiol. Chem. 1900; 31: 543-555Crossref Scopus (10) Google Scholar) respectively, thus making them the two earliest known phosphoproteins. Intriguingly, both these phosphoproteins are secreted from cells. Casein is secreted in milk (3Meggio F. Boulton A.P. Marchiori F. Borin G. Lennon D.P. Calderan A. Pinna L.A. Substrate-specificity determinants for a membrane-bound casein kinase of lactating mammary gland. A study with synthetic peptides.Eur. J. Biochem. 1988; 177: 281-284Crossref PubMed Google Scholar) while phosvitin, a cleaved form of vitellogenin, is synthesized in the liver and secreted into the oviduct (4Byrne B.M. van het Schip A.D. van de Klundert J.A. Arnberg A.C. Gruber M. Ab G. Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene.Biochemistry. 1984; 23: 4275-4279Crossref PubMed Google Scholar, 5Cozza G. Moro E. Black M. Marin O. Salvi M. Venerando A. Tagliabracci V.S. Pinna L.A. The Golgi 'casein kinase' Fam20C is a genuine 'phosvitin kinase' and phosphorylates polyserine stretches devoid of the canonical consensus.FEBS J. 2018; 285: 4674-4683Crossref PubMed Scopus (8) Google Scholar). Since these initial discoveries, casein and phosvitin have been used as common artificial substrates in the study of numerous kinases (6Burnett G. Kennedy E.P. The enzymatic phosphorylation of proteins.J. Biol. Chem. 1954; 211: 969-980Abstract Full Text PDF PubMed Google Scholar, 7Rabinowitz M. Lipmann F. Reversible phosphate transfer between yolk phosphoprotein and adenosine triphosphate.J. Biol. Chem. 1960; 235: 1043-1050Abstract Full Text PDF PubMed Google Scholar, 8Rodnight R. Lavin B.E. Phosvitin kinase from brain: Activation by ions and subcellular distribution.Biochem. J. 1964; 93: 84-91Crossref PubMed Google Scholar). In fact, the first evidence for the existence of protein kinases was provided by the pioneering study of George Burnett and Eugene Kennedy where they used rat mitochondrial extract to provide ATP and casein as the substrate to demonstrate the covalent addition of phosphate to casein in vitro (6Burnett G. Kennedy E.P. The enzymatic phosphorylation of proteins.J. Biol. Chem. 1954; 211: 969-980Abstract Full Text PDF PubMed Google Scholar). Since that time, many investigators have added to the number and complexity of kinases leading to the compilation of the kinome in 2002 (9Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (5717) Google Scholar). This list of the human kinome included 540 individual members and represented kinases that could phosphorylate proteins as well as other biological molecules such as lipids and carbohydrates primarily within the cytosol and nucleus of the cell. But what about the kinases that phosphorylate resident proteins in the secretory pathway or proteins destined for secretion? This question was partially answered when the physiological secretory pathway kinase phosphorylating casein, family of sequence similarity 20C (Fam20C), was discovered in 2012 (10Ishikawa H.O. Xu A. Ogura E. Manning G. Irvine K.D. The Raine syndrome protein FAM20C is a Golgi kinase that phosphorylates bio-mineralization proteins.PLoS One. 2012; 7e42988Crossref PubMed Scopus (109) Google Scholar, 11Tagliabracci V.S. Engel J.L. Wen J. Wiley S.E. Worby C.A. Kinch L.N. Xiao J. Grishin N.V. Dixon J.E. Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.Science. 2012; 336: 1150Crossref PubMed Scopus (280) Google Scholar). This same kinase was found to phosphorylate phosvitin in 2018 and thereby is accountable for the phosphorylation of the first identified secreted phosphoproteins (5Cozza G. Moro E. Black M. Marin O. Salvi M. Venerando A. Tagliabracci V.S. Pinna L.A. The Golgi 'casein kinase' Fam20C is a genuine 'phosvitin kinase' and phosphorylates polyserine stretches devoid of the canonical consensus.FEBS J. 2018; 285: 4674-4683Crossref PubMed Scopus (8) Google Scholar). The first clue for recognizing the secretory pathway kinases came from the identification of the Drosophila protein, four-jointed (Fj), as a secretory pathway kinase that phosphorylated the extracellular domains of atypical cadherins (12Ishikawa H.O. Takeuchi H. Haltiwanger R.S. Irvine K.D. Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains.Science. 2008; 321: 401-404Crossref PubMed Scopus (175) Google Scholar). Using Fj as a BLAST query revealed a small family of related proteins that included Fam20A, B, and C (11Tagliabracci V.S. Engel J.L. Wen J. Wiley S.E. Worby C.A. Kinch L.N. Xiao J. Grishin N.V. Dixon J.E. Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.Science. 2012; 336: 1150Crossref PubMed Scopus (280) Google Scholar). Since little was known about these proteins, they were designated “Fams” based on shared but limited sequence similarity. They all harbor a signal peptide that would direct them into the secretory pathway, but due to a lack of sequence similarity with canonical kinases, none of these atypical kinases were represented in the human kinome. The other domain these proteins share, which is also the sequence of highest homology, is the C-terminal Fam20 domain. Unexpectedly, the conserved Fam20 domain in each of these proteins has a very different function. Fam20C is the Golgi casein kinase responsible for phosphorylating secreted proteins on SxE/pS motifs (11Tagliabracci V.S. Engel J.L. Wen J. Wiley S.E. Worby C.A. Kinch L.N. Xiao J. Grishin N.V. Dixon J.E. Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.Science. 2012; 336: 1150Crossref PubMed Scopus (280) Google Scholar). Fam20A is a pseudokinase that interacts with Fam20C and increases its activity (13Cui J. Xiao J. Tagliabracci V.S. Wen J. Rahdar M. Dixon J.E. A secretory kinase complex regulates extracellular protein phosphorylation.Elife. 2015; 4e06120Crossref PubMed Scopus (0) Google Scholar), and Fam20B is a xylose kinase involved in proteoglycan biosynthesis (14Wen J. Xiao J. Rahdar M. Choudhury B.P. Cui J. Taylor G.S. Esko J.D. Dixon J.E. Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 15723-15728Crossref PubMed Scopus (60) Google Scholar, 15Koike T. Izumikawa T. Tamura J. Kitagawa H. FAM20B is a kinase that phosphorylates xylose in the glycosaminoglycan-protein linkage region.Biochem. J. 2009; 421: 157-162Crossref PubMed Scopus (94) Google Scholar). Over the past few decades, multiple proteins in the extracellular and secretory space have been found to be phosphorylated. Many of these phospho-proteins are secreted into milk, serum, plasma, and cerebrospinal fluid (reviewed in (16Tagliabracci V.S. Xiao J. Dixon J.E. Phosphorylation of substrates destined for secretion by the Fam20 kinases.Biochem. Soc. Trans. 2013; 41: 1061-1065Crossref PubMed Scopus (0) Google Scholar)) and have defined roles in diverse cellular processes from signaling, coagulation, migration, extracellular matrix formation, proteolysis, and biomineralization. The majority of these secreted proteins exhibit a phospho-motif of SxE/pS but to date, we have limited knowledge of the function of the majority of these extracellular phosphorylation events (reviewed in (16Tagliabracci V.S. Xiao J. Dixon J.E. Phosphorylation of substrates destined for secretion by the Fam20 kinases.Biochem. Soc. Trans. 2013; 41: 1061-1065Crossref PubMed Scopus (0) Google Scholar)). Interestingly, out of the 540 kinases in the human kinome, only two kinases have been found localized in the secretory pathway: protein O-mannosyl kinase (POMK/SGK196) (17Zhu Q. Venzke D. Walimbe A.S. Anderson M.E. Fu Q. Kinch L.N. Wang W. Chen X. Grishin N.V. Huang N. Yu L. Dixon J.E. Campbell K.P. Xiao J. Structure of protein O-mannose kinase reveals a unique active site architecture.Elife. 2016; 5e22238Crossref PubMed Scopus (19) Google Scholar, 18Yoshida-Moriguchi T. Willer T. Anderson M.E. Venzke D. Whyte T. Muntoni F. Lee H. Nelson S.F. Yu L. Campbell K.P. SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function.Science. 2013; 341: 896-899Crossref PubMed Scopus (139) Google Scholar) and the tyrosine kinase, vertebrate lonesome kinase (VLK/SGK493) (19Bordoli M.R. Yum J. Breitkopf S.B. Thon J.N. Italiano Jr., J.E. Xiao J. Worby C. Wong S.K. Lin G. Edenius M. Keller T.L. Asara J.M. Dixon J.E. Yeo C.Y. Whitman M. A secreted tyrosine kinase acts in the extracellular environment.Cell. 2014; 158: 1033-1044Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), both of which do not phosphorylate SxE/pS motifs. Because the identity of the kinase(s) responsible for the majority of extracellular phosphorylation events remained elusive, the study of extracellular phosphorylation has lagged behind that of intracellular phosphorylation. It is increasingly clear that extracellular phosphorylation events play just as important roles in cellular regulation as their intracellular counterparts. To date, there are 13 known secretory pathway kinases (or kinase-like proteins), and we know very little about some of them. In a handful of cases, we do not know their substrate specificity or even if they are active kinases. This review focuses on Fam20A, B, C, the small subfamily of secretory pathway kinases for which we have made significant progress. In particular, we will address their cellular functions, reported substrates, structure/function relationships, and importance in human disease. VLK and POMK are two secreted kinases that can be found at the root of the kinome tree. Therefore, their amino acid sequences were well enough conserved with the canonical kinases for them to be classified as kinases. POMK is an O-mannose kinase important for dystroglycan receptor function and matriglycan elongation (18Yoshida-Moriguchi T. Willer T. Anderson M.E. Venzke D. Whyte T. Muntoni F. Lee H. Nelson S.F. Yu L. Campbell K.P. SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function.Science. 2013; 341: 896-899Crossref PubMed Scopus (139) Google Scholar, 20Walimbe A.S. Okuma H. Joseph S. Yang T. Yonekawa T. Hord J.M. Venzke D. Anderson M.E. Torelli S. Manzur A. Devereaux M. Cuellar M. Prouty S. Ocampo Landa S. Yu L. et al.POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan.Elife. 2020; 9e61388Crossref PubMed Google Scholar). VLK is the first secreted tyrosine kinase identified, and it phosphorylates a broad range of secreted and ER-resident substrates (19Bordoli M.R. Yum J. Breitkopf S.B. Thon J.N. Italiano Jr., J.E. Xiao J. Worby C. Wong S.K. Lin G. Edenius M. Keller T.L. Asara J.M. Dixon J.E. Yeo C.Y. Whitman M. A secreted tyrosine kinase acts in the extracellular environment.Cell. 2014; 158: 1033-1044Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). A PSI-BLAST search using VLK as a query produces another small family of potential secreted kinases that includes Fam69A, Fam69B, Fam69C, DIA1, and DIA1R. Very little is known about these proteins (21Hareza A. Bakun M. Świderska B. Dudkiewicz M. Koscielny A. Bajur A. Jaworski J. Dadlez M. Pawłowski K. Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58.PeerJ. 2018; 6e4599Crossref PubMed Scopus (2) Google Scholar, 22Dudkiewicz M. Lenart A. Pawłowski K. A novel predicted calcium-regulated kinase family implicated in neurological disorders.PLoS One. 2013; 8e66427Crossref PubMed Scopus (0) Google Scholar, 23Tennant-Eyles A.J. Moffitt H. Whitehouse C.A. Roberts R.G. Characterisation of the FAM69 family of cysteine-rich endoplasmic reticulum proteins.Biochem. Biophys. Res. Commun. 2011; 406: 471-477Crossref PubMed Scopus (0) Google Scholar). As alluded to in the introduction, the study of extracellular kinases was spearheaded by Ken Irvine’s laboratory when they published the first example of a secreted kinase, the fly protein Fj, which they went on to show phosphorylated unusual cadherin domains (12Ishikawa H.O. Takeuchi H. Haltiwanger R.S. Irvine K.D. Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains.Science. 2008; 321: 401-404Crossref PubMed Scopus (175) Google Scholar). The murine equivalent of Fj, four-jointed box 1 (FJX1) is involved in forming appropriate dendrite arbor morphology in the hippocampus (24Probst B. Rock R. Gessler M. Vortkamp A. Püschel A.W. The rodent four-jointed ortholog Fjx1 regulates dendrite extension.Dev. Biol. 2007; 312: 461-470Crossref PubMed Scopus (29) Google Scholar), and recently, human FJX1 has been shown to increase the invasive potential of nasopharyngeal cancer cells (25Chai S.J. Ahmad Zabidi M.M. Gan S.P. Rajadurai P. Lim P.V.H. Ng C.C. Yap L.F. Teo S.H. Lim K.P. Patel V. Cheong S.C. An oncogenic role for four-jointed box 1 (FJX1) in nasopharyngeal carcinoma.Dis. Markers. 2019; 2019: 3857853Crossref PubMed Scopus (3) Google Scholar, 26Chai S.J. Yap Y.Y. Foo Y.C. Yap L.F. Ponniah S. Teo S.H. Cheong S.C. Patel V. Lim K.P. Identification of four-jointed box 1 (FJX1)-specific peptides for immunotherapy of nasopharyngeal carcinoma.PLoS One. 2015; 10e0130464Crossref PubMed Scopus (6) Google Scholar). In addition to FJX1 and Fam20A, B, and C, this small family contains two additional members, Fam198A and Fam198B. To date, neither Fam198A nor B has been ascribed kinase activity, and very little is known about their cellular functions (27Hsu C.Y. Chang G.C. Chen Y.J. Hsu Y.C. Hsiao Y.J. Su K.Y. Chen H.Y. Lin C.Y. Chen J.S. Chen Y.J. Hong Q.S. Ku W.H. Wu C.Y. Ho B.C. Chiang C.C. et al.FAM198B is associated with prolonged survival and inhibits metastasis in lung adenocarcinoma via blockage of ERK-mediated MMP-1 expression.Clin. Cancer Res. 2018; 24: 916-926Crossref PubMed Scopus (9) Google Scholar, 28Wei Z. Liu T. Lei J. Wu Y. Wang S. Liao K. Fam198a, a member of secreted kinase, secrets through caveolae biogenesis pathway.Acta Biochim. Biophys. Sin. (Shanghai). 2018; 50: 968-975Crossref PubMed Scopus (2) Google Scholar). Vertebrates exhibit three members of the Fam20 family of proteins (Fam20A,B, and C), whereas early invertebrates such as hydra and sponge have a single homolog of Fam20 whose activity resembles the human Fam20B-like protein (Fig. 1) (29Zhang H. Zhu Q. Cui J. Wang Y. Chen M.J. Guo X. Tagliabracci V.S. Dixon J.E. Xiao J. Structure and evolution of the Fam20 kinases.Nat. Commun. 2018; 9: 1218Crossref PubMed Scopus (27) Google Scholar). Within the Fam20 family of secretory kinases, Fam20B was identified as a xylosylkinase kinase that phosphorylates xylose residues within the conserved tetrasaccharide linkages of proteoglycans (15Koike T. Izumikawa T. Tamura J. Kitagawa H. FAM20B is a kinase that phosphorylates xylose in the glycosaminoglycan-protein linkage region.Biochem. J. 2009; 421: 157-162Crossref PubMed Scopus (94) Google Scholar). Interestingly, the xylose phosphorylation on the proteoglycan tetrasaccharide linkage was first identified in hydra (30Yamada S. Morimoto H. Fujisawa T. Sugahara K. Glycosaminoglycans in Hydra magnipapillata (Hydrozoa, Cnidaria): Demonstration of chondroitin in the developing nematocyst, the sting organelle, and structural characterization of glycosaminoglycans.Glycobiology. 2007; 17: 886-894Crossref PubMed Scopus (41) Google Scholar), and further biochemical investigation revealed that hydra Fam20 and sponge Fam20 lacked protein kinase activity but exhibited robust xylosylkinase activity (29Zhang H. Zhu Q. Cui J. Wang Y. Chen M.J. Guo X. Tagliabracci V.S. Dixon J.E. Xiao J. Structure and evolution of the Fam20 kinases.Nat. Commun. 2018; 9: 1218Crossref PubMed Scopus (27) Google Scholar). In fact, Fam20B is thought to be the first ancestral template protein for the Fam20 family of kinases and the function of xylose phosphorylation is conserved through the animal phylum from sponges to humans (29Zhang H. Zhu Q. Cui J. Wang Y. Chen M.J. Guo X. Tagliabracci V.S. Dixon J.E. Xiao J. Structure and evolution of the Fam20 kinases.Nat. Commun. 2018; 9: 1218Crossref PubMed Scopus (27) Google Scholar). This evolutionary relationship is apparent in available structures. The ATP-binding sites of Fam20B and Fam20C are highly conserved (Fig. 2, A and B). However, Fam20B has a unique saccharide binding site not present in Fam20C or Fam20A (Fig. 2, A and C) (29Zhang H. Zhu Q. Cui J. Wang Y. Chen M.J. Guo X. Tagliabracci V.S. Dixon J.E. Xiao J. Structure and evolution of the Fam20 kinases.Nat. Commun. 2018; 9: 1218Crossref PubMed Scopus (27) Google Scholar). Fam20C homologs are characterized by an occluded substrate binding pocket that cannot accommodate bulky saccharide substrate due to steric clashes. This occlusion results from slight structural rearrangements arising from distal residue substitutions that position a flexible loop within the binding pocket (Fig. 2D) (29Zhang H. Zhu Q. Cui J. Wang Y. Chen M.J. Guo X. Tagliabracci V.S. Dixon J.E. Xiao J. Structure and evolution of the Fam20 kinases.Nat. Commun. 2018; 9: 1218Crossref PubMed Scopus (27) Google Scholar). The Fam20B-mediated xylose phosphorylation robustly stimulates galactosyltransferase II (GalT-II) activity leading to further addition of galactose to the tetrasaccharide linkages and accelerated proteoglycan chain extension (Fig. 5) (14Wen J. Xiao J. Rahdar M. Choudhury B.P. Cui J. Taylor G.S. Esko J.D. Dixon J.E. Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 15723-15728Crossref PubMed Scopus (60) Google Scholar). Furthermore, EXTL2 (Exostosin-Like Glycosyltransferase 2) polymerase utilizes the xylose phosphorylation to transfer a GlcNAc residue to the tetrasaccharide linkage region leading to termination of proteoglycan chain elongation (31Nadanaka S. Zhou S. Kagiyama S. Shoji N. Sugahara K. Sugihara K. Asano M. Kitagawa H. EXTL2, a member of the EXT family of tumor suppressors, controls glycosaminoglycan biosynthesis in a xylose kinase-dependent manner.J. Biol. Chem. 2013; 288: 9321-9333Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Intriguingly, depletion of Fam20B leads to immature proteoglycan formation, a phenotype quite reminiscent of Ehlers–Danlos syndrome, a rare inherited condition that affects connective tissue owing to GalT-II mutations (14Wen J. Xiao J. Rahdar M. Choudhury B.P. Cui J. Taylor G.S. Esko J.D. Dixon J.E. Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 15723-15728Crossref PubMed Scopus (60) Google Scholar). Thus, Fam20B plays an evolutionarily conserved quality-control role for proteoglycan biosynthesis and is arguably the ancestral Fam20.Figure 2Structure of FAM20B, the glycan kinase. A, structure of Hydra magnipapillata FAM20B (hmFAM20B, PDB ID: 5xoo, chain A, white) with bound adenosine (ADN) and Galβ1-4Xylβ1 substrate. N and C lobes indicated approximately. B, FAM20B ATP-binding site (PDB ID:5xoo, chain A, white, ADN:adenosine) is highly conserved with C. elegans FAM20C ATP-binding site (PDB ID:4kqb, chain A, goldenrod, ADP, adenosine diphosphate). Similar residues labeled (FAM20B:black, FAM20C:orange). C, FAM20B saccharide binding site containing Galβ1-4Xylβ1 substrate (gray) (PDB ID:5xoo, chain A). D, superimposed FAM20B (PDB ID:5xoo, chain A, white) with C. elegans FAM20C (PDB ID:4kqb, chain A) at saccharide binding site. Arrow indicates flexible loop occluding saccharide binding. E, gene diagram depicting disease mutations. fs, frame shift.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Whole-body genetic depletion of Fam20B in mice was embryonic lethal at E13.5 with the embryos exhibiting severe development defects and significant organ hypoplasia (32Vogel P. Hansen G.M. Read R.W. Vance R.B. Thiel M. Liu J. Wronski T.J. Smith D.D. Jeter-Jones S. Brommage R. Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.Vet. Pathol. 2012; 49: 998-1017Crossref PubMed Scopus (0) Google Scholar). These observations were consistent with studies in zebrafish wherein loss-of-function mutants of Fam20B led to aberrant cartilage matrix organization and early stages of chondrocyte hypertrophy leading to skeletal defects (33Eames B.F. Yan Y.L. Swartz M.E. Levic D.S. Knapik E.W. Postlethwait J.H. Kimmel C.B. Mutations in fam20b and xylt1 reveal that cartilage matrix controls timing of endochondral ossification by inhibiting chondrocyte maturation.PLoS Genet. 2011; 7e1002246Crossref PubMed Scopus (75) Google Scholar). These initial in vivo observations were further echoed when tissue-specific depletion of Fam20B in mice led to the development of supernumerary teeth (34Tian Y. Ma P. Liu C. Yang X. Crawford D.M. Yan W. Bai D. Qin C. Wang X. Inactivation of Fam20B in the dental epithelium of mice leads to supernumerary incisors.Eur. J. Oral Sci. 2015; 123: 396-402Crossref PubMed Scopus (13) Google Scholar, 35Wu J. Tian Y. Han L. Liu C. Sun T. Li L. Yu Y. Lamichhane B. D'Souza R.N. Millar S.E. Krumlauf R. Ornitz D.M. Feng J.Q. Klein O. Zhao H. et al.FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling.BMC Biol. 2020; 18: 87Crossref PubMed Scopus (0) Google Scholar), chondrosarcoma with major postnatal ossification defects (36Ma P. Yan W. Tian Y. Wang J. Feng J.Q. Qin C. Cheng Y.S. Wang X. Inactivation of Fam20B in joint cartilage leads to chondrosarcoma and postnatal ossification defects.Sci. Rep. 2016; 6: 29814Crossref PubMed Scopus (8) Google Scholar), and severe craniofacial defects (37Liu X. Li N. Zhang H. Liu J. Zhou N. Ran C. Chen X. Lu Y. Wang X. Qin C. Xiao J. Liu C. Inactivation of Fam20b in the neural crest-derived mesenchyme of mouse causes multiple craniofacial defects.Eur. J. Oral Sci. 2018; 126: 433-436Crossref PubMed Scopus (4) Google Scholar). Thus, the overarching role of Fam20B in proteoglycan biosynthesis likely contributes to the skeletal and developmental defects observed upon Fam20B depletion in tissue-specific in vivo models. In humans, two lethal compound heterozygous variants in Fam20B have been identified in a girl who died soon after birth (Fig. 2E) (38Kuroda Y. Murakami H. Enomoto Y. Tsurusaki Y. Takahashi K. Mitsuzuka K. Ishimoto H. Nishimura G. Kurosawa K. A novel gene (FAM20B encoding glycosaminoglycan xylosylkinase) for neonatal short limb dysplasia resembling Desbuquois dysplasia.Clin. Genet. 2019; 95: 713-717Crossref PubMed Scopus (0) Google Scholar). The genetic alterations reported were T59Afs and N347Mfs and the patient exhibited severe organ hypoplasia, skeletal defects, and respiratory failure (38Kuroda Y. Murakami H. Enomoto Y. Tsurusaki Y. Takahashi K. Mitsuzuka K. Ishimoto H. Nishimura G. Kurosawa K. A novel gene (FAM20B encoding glycosaminoglycan xylosylkinase) for neonatal short limb dysplasia resembling Desbuquois dysplasia.Clin. Genet. 2019; 95: 713-717Crossref PubMed Scopus (0) Google Scholar). The amino terminal T59A frameshift leads to hypomorphic gene function and essential loss of one allele of Fam20B. The carboxy-terminal alteration, N347M frameshift, results in disruption of more than 15% of the protein sequence and results in the loss of C389, which forms a disulfide bond with C332 and likely contributes to the global stability of the protein. The N347M frameshift, therefore, results in a destabilized Fam20B and also represents a functionally inactive variant. Intriguingly, osteoarthiritis and osteochondropathy patients with decreased proteoglycans and chondrocyte numbers exhibited marked reduction of Fam20B, GalT-II, and EXTL2 protein levels in knee cartilage biopsy samples (39Lei J. Deng H. Ran Y. Lv Y. Amhare A.F. Wang L. Guo X. Han J. Lammi M.J. Altered expression of aggrecan, FAM20B, B3GALT6, and EXTL2 in patients with osteoarthritis and Kashin-beck disease.Cartilage. 2020; https://doi.org/10.1177/1947603520932199Crossref PubMed Scopus (1) Google Scholar). This suggests that Fam20B could be a predictive marker for specific bone diseases. As stated previously, the story of milk casein as a phosphoprotein started in the late 19th century when Olof Hammarsten reported the presence of phosphorus in casein (1Hammarsten O. Zur Frage ob das Caseïn ein einheitlicher Stoff sei.Z. Physiol. Chem. 1883; 7: 227-273Google Scholar). Fifty years later, Fritz Lipmann identified that the phosphorus was covalently bound to casein as phosphoseryl groups (40Lipmann F. Über die Bindung der Phosphorsäure in Phosphorproteinen. I.Biochem. Z. 1933; 262: 3-8Google Scholar). Eventually, the sequences surrounding those phosphoseryl groups in casein were identified as SxE/pS, which prompted the idea that SxE/pS sequence was the preferential motif for enzymes phosphorylating casein (41Mercier J.C. Phosphorylation of caseins, present evidence for an amino-acid triplet code post-translationally recognized by specific kinases.Biochimie. 1981; 63: 1-17Crossref PubMed Scopus (0) Google Scholar, 42Mercier J.C. Grosclaude F. Ribadeau-Dumas B. 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