Critical Role of the Potential O-Linked Glycosylation Sites of CXCR4 in Cell Migration and Bone Marrow Homing of Hematopoietic Stem Progenitor Cells

Hematology Immunology Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine
CXCR4 O-linked glycosylation hematopoietic stem progenitor cells cell migration bone marrow homing glycosylation CXCL12

1. Academic Background and Research Origin

Hematopoietic stem/progenitor cells (HSPCs) are the foundation for maintaining the steady state of the adult blood system. Every day, the human body needs to generate billions of new blood cells—a process that relies on the capacity of HSPCs for self-renewal and directed differentiation within the bone marrow microenvironment. Bone marrow transplantation (BMT) has long been adopted as a treatment for certain hematologic diseases (such as aplastic anemia, hemophilia, multiple myeloma, etc.). Successful transplantation requires HSPCs to efficiently “home” to, and firmly engraft in, the recipient’s bone marrow, serving as the source for rebuilding the blood cell lineages.

During the homing process, interactions between HSPCs and adhesion molecules in the bone marrow microenvironment (e.g., VCAM-1, ICAM-1), integrins (e.g., VLA-4, VLA-5), and chemokines play precise regulatory roles. Among them, the C-X-C chemokine receptor 4 (CXCR4) and its ligand C-X-C ligand 12 (CXCL12, also known as SDF-1) are recognized as the molecular axis of HSPC homing. Previous research has confirmed that deletion of CXCR4 leads to embryonic lethality and defects in hematopoiesis and heart development, while overexpressing CXCR4 can enhance the homing capacity of human CD34+ cells.

In addition, cell surface glycan modifications (glycosylation) are fundamental in cell–cell recognition, migration, and adhesion. The main types of glycosylation are N-linked and O-linked glycosylation. Prior work from the research team already found that glycosylation defects (such as β-1,4-galactosyltransferase 1 deficiency) reduce the efficiency of HSPC homing. However, whether glycosylation of the CXCR4 molecule specifically contributes to HSPC homing, and which particular sites are involved in relevant regulatory mechanisms, remain unclear. Therefore, this study aims to reveal the mechanistic role of O-linked glycosylation sites on the CXCR4 molecule in bone marrow homing and migration of HSPCs, thus providing theoretical foundations and potential clinical tools for optimizing hematopoietic stem cell transplantation and screening suitable cells.

2. Source of the Paper and Author Introduction

This study was conducted by Xuchi Pan, Chie Naruse (corresponding author), Tomoko Matsuzaki, Ojiro Ishibashi, Kazushi Sugihara, Hidetsugu Asada, and Masahide Asano (corresponding author), all from the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, and its affiliated institutions in Japan. The paper was published on May 4, 2025, by Oxford University Press, included in the journal Stem Cells (stem cells, 2025, vol. 43, no. 6, sxaf025), and is an original academic article focusing on basic and translational research.

3. Detailed Description of the Overall Research Process

1. Research Design Overview

The study centered on O-linked glycosylation site prediction at specific regions of the CXCR4 N-terminus, and, through point mutation and gene editing, systematically evaluated the functional impact of these sites on cell migration and homing. Experiments spanned human HEK293, mouse NIH/3T3 cell lines, and primary mouse hematopoietic stem/progenitor cells. Combined with genetically engineered mouse models, the study established a comprehensive experimental system covering cellular function, molecular mechanisms, in vivo homing, and reconstitution in recipients.

1.1 Glycosylation Site Prediction and Gene Mutagenesis

  • The study first utilized NetOGlyc 4.0 to predict O-linked glycosylation sites within the N-terminal 1-38 amino acids of mouse CXCR4, identifying Ser-5, Ser-9, and Ser-18 as potential sites, with a focus on Ser-5 and Ser-9.
  • Based on this, the team used a point mutation method (substituting serine with alanine, S5A/S9A) to construct various CXCR4 mutants, including CXCR4S5A, CXCR4S9A, CXCR4S5AS9A, CXCR4N11Q (N-linked glycosylation–related mutant), and CXCR4S5ASN11Q, among others.

1.2 Cell Functional Experiments

  • Lentiviral (LV) vectors were used to overexpress wild-type and mutant CXCR4 proteins in HEK293 and NIH/3T3 cells. After antibiotic selection, clones exhibiting similar expression levels were chosen for further experiments.
  • Cell migration capacity was assessed using wound healing assays (to test non-directional migration) and Transwell migration assays (to test chemotactic migration).
  • In some experiments, the CXCR4 antagonist AMD3100 was used to validate migration dependence.

1.3 Molecular Mechanism Investigations

  • Comparison of membrane expression and molecular weight differences between wild-type and mutant CXCR4 assessed the effects of glycosylation on molecular size and stability.
  • Lectin blotting using different lectins (Jacalin, PNA, SNA, etc.) was performed to probe structural features of O-linked glycans (such as sialic acid linkages).
  • Flow cytometry (FACS) analyzed differences in binding affinity between CXCR4 and CXCL12, and Western blotting assessed activation levels of canonical downstream pathways such as FAK, MEK1/2, and PI3K.

1.4 Mouse HSPC Homing and Transplantation Function Evaluation

  • CRISPR/Cas9 was used to generate CXCR4−/−, CXCR4S5A/S5A, CXCR4S9A/S9A, and CXCR4S5AS9A/S5AS9A mutant mice.
  • Kit+ hematopoietic progenitor cells were harvested from fetal liver or adult mouse bone marrow, purified by magnetic bead sorting and cultured in vitro. Cells of different genotypes were then transplanted into recipient mice lethally irradiated by γ-rays.
  • Recipient bone marrow was collected 24 hours later, and HSPC homing was quantified using flow cytometry. Post-transplant survival and long-term hematopoietic reconstitution were also monitored.

2. Detailed Major Experimental Results and Significance

2.1 Role of CXCR4 O-linked Glycosylation Sites in Cell Migration

  • Wound healing and Transwell assays showed that overexpression of wild-type CXCR4 significantly enhanced cell migration toward CXCL12, and this effect could be completely blocked by AMD3100.
  • Single-point mutants (S5A or S9A) or N-linked glycosylation mutants (N11Q) had migration abilities similar to wild-type. However, double-point mutants (S5AS9A) completely lost migration response to CXCL12. Multiple-point mutants (S5AS9AN11QS18A) also lost function, highlighting the critical roles of the O-linked glycosylation residues S5 and S9.

2.2 Glycosylation at Ser-5/Ser-9 and Supporting Molecular Evidence

  • Western blot showed that loss of N-linked glycosylation (N11Q) reduced the molecular weight of CXCR4, and S5AS9A double mutation further lowered it.
  • Lectin blotting demonstrated that PNA and Jacalin binding was significantly weakened for the S5AS9AN11Q mutant, especially after sialidase treatment. This suggests that the Ser-5/Ser-9 sites are linked with O-linked glycans such as Galβ1,3GalNAc, and terminated with α2,6-linked sialic acid.
  • Attempts at further glycan structure elucidation using LC/MS/MS failed due to instability of CXCR4 protein and unsuccessful purification/peptide identification.

2.3 Receptor Binding and Downstream Signal Transduction

  • Flow cytometry confirmed that the S5AS9A double mutant significantly reduced CXCR4 binding to anti-CXCL12 antibody. Wild-type CXCR4 reached peak CXCL12 binding at 2 hours, while the mutant’s response was markedly lower and weaker.
  • Western blotting showed that wild-type CXCR4 activated migration pathways FAK, MEK1/2, and PI3K in response to CXCL12, while S5AS9A mutants were completely inactive, consistent in both kinetics and phenotype.

2.4 HSPC Homing and Transplantation Reconstitution

  • LV-infected CXCR4−/− kit+ fetal liver cells expressing empty vector, wild-type, or S5AS9A mutant CXCR4 were transplanted into recipients. Only wild-type CXCR4 restored homing function; the mutant showed no improvement.
  • Kit+ fetal liver or bone marrow cells from CRISPR-edited CXCR4S5AS9A/S5AS9A mice had homing efficiencies reduced to about 50% of CXCR4+/− and similar to CXCR4−/−. Single mutants behaved similarly to wild-type.
  • Following transplantation of different doses of kit+ fetal liver cells, recipient mouse survival rate decreased in line with homing efficiency. S5AS9A/S5AS9A and CXCR4−/− were comparable: high doses (1×10^5 cells) rescued recipients, but medium/low doses resulted in significantly lower survival rates, showing statistical differences from wild-type and heterozygotes.

3. Results Chain and Reasoning to Conclusions

  • O-linked glycosylation residues Ser-5 and Ser-9 are crucial for CXCR4-mediated HSPC migration, dramatically affecting CXCR4—CXCL12 binding, downstream signaling activation, and ultimately HSPC homing efficiency.
  • Mouse models generated via CRISPR confirmed that complete loss of glycosylation at these sites (S5AS9A/S5AS9A) leads to impaired hematopoietic reconstitution ability and reduced bone marrow transplantation survival rates, offering valuable guidance for clinical stem cell transplantation screening.

4. Scientific and Applied Value

This study is the first to systematically reveal that Ser-5 and Ser-9 O-linked glycosylation of CXCR4 is a key molecular mechanism regulating HSPC migration and homing. It provides novel evidence at the cellular and molecular level for the significant differences in HSPC homing and reconstitution efficiency between individuals. The finding suggests that clinical problems such as poor homing during cord blood transplantation may be partially due to abnormal glycosylation of CXCR4. Utilization of lectin and similar molecular tools to screen HSPCs with normal glycosylation may, in the future, optimize transplant matching and therapeutic effectiveness.

5. Research Features and Highlights

  • Discovery of the independent role of O-linked glycosylation (Ser-5, Ser-9) on CXCR4 in HSPC migration and homing, elucidating a novel layer of regulation by cell-surface glycosylation on the chemokine receptor–ligand system.
  • Fine mutant mouse models constructed via CRISPR/Cas9, systematically dissecting the effect of point mutations on hematopoietic stem/progenitor cell function and transplantation reconstitution, setting an example for molecular precision medicine.
  • First to propose screening the glycosylation state of clinical HSPCs using lectins, opening new avenues for optimizing the sources of HSPCs and improving transplantation success rates.

6. Research Limitations and Future Directions

The authors point out that current techniques cannot fully exclude the direct effect of point mutations on molecular function, and adequate amounts of complete CXCR4 have not yet been purified to resolve the precise glycan structures. In the future, the team plans to further optimize protein expression and purification, coupled with high-resolution proteomics for direct evidence. Moreover, due to resource and technical limitations, studies on human HSPCs have not yet been initiated; further human cell experimentation would enhance clinical translatability.

4. Summary and Academic Impact

This study, conducted by the Institute of Laboratory Animals, Kyoto University, and others, was published in Stem Cells in 2025. Through molecular biology, cell functional studies, and in vivo transplantation models, the work systematically demonstrates the irreplaceable role of O-linked glycosylation modification at Ser-5 and Ser-9 of the CXCR4 receptor on hematopoietic stem/progenitor cell surfaces in cell migration, bone marrow homing, and transplantation reconstitution. This discovery not only enriches the molecular landscape of HSPC migration regulation but also provides new theoretical and technical directions for cell selection and effect improvement in clinical transplantation. Future translation of the relevant technologies may help solve the clinical issue of inefficient HSPC homing during transplantation and promote advances in regenerative medicine and precision transplantation medicine.